Metal radionuclide labeled proteins for diagnosis and therapy

ABSTRACT

Protein conjugated chelated metal radionuclides are provided for use in vivo. Intermediates are provided for preparing the polypeptide compositions efficiently.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.817,321, filed Jan. 9, 1986, which is a continuation-in-part ofapplication Ser. No. 692,000, filed Jan. 14, 1985.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Radiolabeled compounds are important tools in medical diagnosis andtreatment. Such compounds are employed in a variety of techniquesincluding the diagnosis of deep venous thrombi, the study of lymph nodepathology, and the detection, staging and treatment of neoplasms. Anumber of these compounds employ metal radionuclides such asTechnetium-99m. When employing radionuclides for in vivo administrationit is desirable that the radionuclide localize in a target organ orcancer site. Therefore, radionuclides are usually formulated to providepreferential binding to or absorption by the particular organ or tissue.There is considerable interest in being able to accurately direct aradionuclide to a preselected site to reduce background radiationdirected to surrounding or distant tissue, reduce the dosage, minimizebackground for in vivo imaging and minimize undesirable side effects.Toward this end, methods involving specific ligands or receptors towhich the radionuclide may be conjugated are of interest.

Publications of interest include Khaw, et al., J. Nucl. Med. (1982)23:1011; Rhodes, B.A., Sem. Nucl. Med. (1974) 4:281; Davidson, et al.,Inorg. Chem. (1981) 20:1629; and Byrne and Tolman, J. Nucl. Med. (1983)24:126. See particularly Fritzberg, et al., J. Nucl. Med. (1982) 23:592:Fritzberg, et al., ibid. (1981) 22:258; and Fritzberg, et al., ibid.(1982) 23:17 for descriptions of mercaptoacetyl derivatives of ethylenediamine carboxylic acid derivatives. See also U.S. Pat. Nos. 4,434,151,4,444,690, and 4,472,509 whose disclosures are incorporated herein byreference.

SUMMARY OF THE INVENTION

Metal radionuclide labeled proteins are provided for the diagnosis andtreatment of a variety of pathologic conditions. Specifically, chelatedradionuclide protein conjugates are employed for the diagnosis ofconditions including lymph node pathology and deep venous thrombi andthe detection and staging of neoplasms. Also, chelated radionuclides asprotein conjugates are employed for radiotherapy of tumors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (a-c) is a flow chart representing the preparation of a ^(99m)Tc-radiolabeled polypeptide using a kit in accordance with oneembodiment of the invention.

FIG. 2 (a-e) is a flow chart representing the preparation of apolypeptide radiolabeled with a rhenium isotope using a kit inaccordance with one embodiment of the invention.

FIGS. 3 (a-b), 4, 5 (a-b), and 6 (a-b) show biodistribution data forvarious antibody fragments radiolabeled with ^(99m) Tc or ¹⁸⁸ Re inaccordance with the invention, and injected into tumor-bearing mice. Theantibody fragments are specific for various types of cancer cells.

DETAILED DESCRIPTION OF THE INVENTION

Improved methods and compositions are provided related to metalradionuclide chelates, their active esters for conjugating to proteins,and the resulting peptide conjugates, as well as the use of theconjugates in radioimaging and radiotherapy.

The metal chelating compounds will be dithio, diaminoordiamidocarboxylic acids or amines or derivatives thereof, e.g., aN,N'-bis-mercaptoacetyl ,(w-x)-diamino carboxylic acid, (x is 1 or 2),esters capable of forming an amide bond in an aqueous medium with apolypeptide, and intermediates to the chelate. The chelating compoundsare referred to as N₂ S₂ ligands or chelates.

The compounds of this invention will for the most part have thefollowing formula: ##STR1## wherein:

one of Z¹, Z², Z³ or Z⁴ is RCW-(HNV)_(n) Y, and the others are═O or H₂ ;

R is a divalent organic radical of at least 1 carbon atom and typicallynot more than about 10, usually not more than 6 carbon atoms, usuallyfrom 1 to 3 carbon atoms having from 0 to 2 heteroatoms which arechalcogen (O, S) or nitrogen and is aliphatic, alicyclic, aromatic orheterocyclic, preferably aliphatic having from 0 to 2, usually 0 to 1site of aliphatic unsaturation (e.g., ethylenic) and of from 1 to 2carbon atoms;

W is oxygen or imino (═O or ═NH), with the proviso that when Y is --NH₂or --NHNH₂, the W bonded to the carbon atom bonded to Y is H₂ ;

V is RCW, where the two RCWs may be the same or different, usually beingof from 1 to 8, more usually of from 1 to 6 carbon atoms, preferably offrom 2 to 3 carbon atoms;

n is 0 or 1;

T is an acyl or acylthio radical of from 2 to 10, usually 2-8 carbonatoms, either a hydrocarbyl acyl or substituted acylradical, usuallyaryl (e.g., phenyl) or alkyl (e.g., methyl), an organic sulfhydrylradical of from 1 to 10 carbon atoms, either substituted orunsubstituted hydrocarbyl; a heterocycle, particularly a chalcogen (O,S) heterocycle; an acylamidomethylene, where the acyl group is asdefined above; hydrogen; sulfonato; an alkali metal ion; or the two T'smay be taken together to define a polyvalent metal radionuclide, as themetal ion or metal ion oxide;

Substituents include nitro, cyano, inert halo (aryl or polyhalo),non-oxo-carbonyl (carboxylic acid, amide and ester), and the like;

Y is hydroxyl, an oxy salt, particularly an alkali metal salt (e.g.,lithium, sodium and potassium), an organic oxy compound forming anester, usually lower alkoxy of from 1 to 6 carbon atoms or a group whichpermits amide formation in an aqueous medium, particularly with apolypeptide, --NH₂, --NHNH₂, or a polypeptide of at least two aminoacids which may be 2 MDal (megadalton) or more. With polypeptides,particularly polypeptides over 1 KDal (kilodalton), there may be morethan one chelating compound bound to the polypeptide, usually not morethan about one per 0.5 KDal;

A's are the same or different and are hydrogen or lower alkyl of from 1to 6 carbon atoms, usually of from 1 to 3 carbon atoms, particularlymethyl, usually hydrogen; and

X is a bond, methylene or CHZ⁴ ;

where T is other than M or H, Y will be other than a polypeptide.

The link between CW and the polypeptide will vary depending upon thenature of CW-Y. Where CW-Y includes a bond formed by reaction with afree amine group on the polypeptide Y, the linkage will be either acarboxamide or amidine depending on whether W is ═O or ═NH. If, however,CW-Y defines a methyleneamine or methylenehydrazine, then reductiveamination may be required with a sugar-substituted-polypeptide which hasbeen cleaved to the oxo group (e.g., glycol cleavage with periodate).Reductive amination may be achieved by combining the oxosubstitutedpolypeptide with the amino- or hydrazino-substituted N₂ S₂ ligand in thepresence of a reducing agent, such as sodium cyanoborohydride.

A preferred group of compounds will have one of the following formulas:##STR2## wherein all of the symbols have been defined previously exceptfor M and T', and wherein:

M is a radionuclide capable of being chelated as the metal ion or metalion oxide; and

T' is a sulfur protective group, which includes acyl, acylthio,hydrocarbylthio or substituted-hydrocarbylthio or heterocyclicthio,where the acyl and hydrocarbyl groups may be aliphatic, alicyclic,aromatic or combinations thereof and the acyl group further includesheterocyclic, wherein acyl is normally carboxyacyl; T' will generally beof from 2 to 10 carbon atoms, usually 2 to 8 carbon atoms when acyl,where substituents will include non-oxo-carbonyl (carboxy), halo (aryl),particularly fluoro and chloro, cyano and nitro.

In one embodiment of the invention, in accordance with this preferredgroup of compounds, one of Z₁ or Z₂ is RCW-(HNV)_(n) Y, and the other is═O or H₂ ; wherein R is the divalent radical --(CH₂)₂ --, W is oxygen(═O), n is 0 and Y is the leaving group of an ester. The other symbolsare as previously defined. Thus, these preferred compounds comprisealiphatic esters of five carbon atoms, and RCW-(HNV)_(n) Y may berepresented as ##STR3## where the other two carbons which are part ofthe 5-carbo comprising the ester group are in the formulae presentedabove. The carbon atom which is β to the carboxyl carbon in the chainmay be substituted with groups other than hydrogen, as long as thereactivity of the ester toward a protein is not diminished throughsteric hindrance. Permissible substituents on this carbon atom include,for example, oxygen (═O) and straight-chain lower alkyl groups (e.g.,methyl and ethyl groups). The chain length of five carbons is preferredbecause it has been found to be long enough to minimize steric hindranceof the reaction of the ester with a polypeptide, yet short enough sothat the chelate compound retains the desirable water solubility(described above). Thus, these preferred compounds generally are morereactive toward polypeptides than are compounds having one or morecarbons deleted from the chain, due to the increasing steric hindrancethat accompanies decreasing chain length. Water solubility has beenfound to decrease as carbons are added to the chain. Increasing chainlength also increases hepatobiliary excretion of the carboxylate form ofthe metal complex. Thus, metabolic release of the complex with longerside chains increases the likelihood of undesirable localization in theliver and subsequently the lower abdomen (gut). These preferredcompounds of 5-carbon chain length may be referred to as C₅ N₂ S₂chelating compounds or chelate compounds.

A group of chelate compounds according to this invention will for themost part have the following formula: ##STR4## wherein:

one of Z¹ ', Z² ', Z³ ' or Z⁴ ' is R'CW'(HNV')_(n) 'Y',and the othersare ═O or H₂ ;

(A')'s are the same or different and are hydrogen or lower alkyl of from1 to 6, usually 1 to 3 carbon atoms, particularly methyl, usuallyhydrogen;

n' is 0 or 1;

V' is R'CW', where the (R'CW')'s may be the same or different, usuallybeing of from 1 to 8, more usually of from 1 to 6 carbon atoms,preferably of from 2 to 3 carbon atoms;

W' is oxygen or imino (═N or ═O), with the proviso that when Y' is ═NH₂or NHNH₂, the W' bonded to the carbon atom bonded to Y is H₂ ;

M is a radionuclide capable of being chelated as the metal ion or metalion oxide;

X' is a bond, methylene or CHZ⁴ ;

R' is an aliphatic divalent radical of from 1 to 6, usually from 1 to 3carbon atoms, having from 0 to 1 site of aliphatic unsaturation and 0 to2 heteroatoms, usually straight chain and preferably methylene orpolymethylene of from 2 to 3 carbon atoms; and

Y' is hydroxyl, an oxy salt, particularly an alkali metal salt, such assodium, an ester of an hydroxylic compound, where the ester is capableof forming an amide bond with a polypeptide in an aqueous medium withoutdenaturation of the polypeptide; --NH_(2;) --NHNH_(2;) an amino acid, ora polypeptide usually of at least about 1000 molecular weight, moreusually at least about 2000 molecular weight, generally less than about1.6 MDal, more usually less than about 800 KDal. Of particular interestare immunoglobulins or specific binding fragments thereof.

The dashed lines in the formulae presented for the chelate compounds ofthe invention represent four coordinate covalent bonds between the metalradionuclide M and each of the two sulfur and the two nitrogen atomsshown in the formulae. Thus, the metal radionuclide is bound throughrelatively stable bonds in the chelate compounds of the invention.

A variety of metals may be employed as the radionuclide. These metalsinclude copper (e.g., ⁶⁷ Cu and ⁶⁴ Cu); technetium (e.g., ^(99m) Tc);rhenium (e.g., ¹⁸⁶ Re and ¹⁸⁸ Re); lead (e.g., ²¹² Pb); bismuth (e.g.,²¹² Bi); and palladium (e.g., ¹⁰⁹ Pd). Methods for preparing theseisotopes are known. Molybdenum/technetium generators for producing^(99m) Tc are commercially available. Procedures for producing ¹⁸⁶ Reinclude the procedures described by Deutsch et al, (Nucl. Med. Biol.Vol. 13:4:465-477, 1986) and Vanderheyden et al. (Inorganic Chemistry,Vol. 24:1666-1673, 1985), and methods for production of ¹⁸⁸ Re have beendescribed by Blachot et al (Intl. J. of Applied Radiation and IsotopesVol. 20:467-470, 1969) and by Klofutar et al (J. of RadioanalyticalChem., Vol. 5:3-10, 1970). Production of ¹⁰⁹ Pd is described in Fawwazet al., J. Nucl. Med. (1984), 25:796. Production of ²¹² Pb and ²¹² Bi isdescribed in Gansow et al., Amer. Chem. Soc. Symp. Ser. (1984),241:215-217, and Kozah et al., Proc. Nat'l. Acad. Sci. USA (January1986) 83:474-478.

The esters are those esters which provide for the reaction with apolypeptide in aqueous medium. One or another of the esters may bepreferred, depending upon the particular radionuclide, the protein andthe conditions for conjugation. As used herein, the term "aqueousmedium" is meant to include not only totally aqueous media but alsomixed aqueous/organic media, wherein the organic component is presentonly in a relatively low concentration, i.e., a concentration low enoughto minimize damage to (e.g., denaturation of) polypeptides in theaqueous medium. A variety of esters may be used, including aromaticesters containing electron-withdrawing groups, or α-substituted methylesters (in which the substituents are electron withdrawing groups, suchas, but not limited to, ##STR5##

Preferred esters for use as Y or Y' groups in the present invention haveseveral structural features which impact the desired stability andreactivity to the esters. For example, preferred esters should berelatively stable, especially with respect to hydrolysis in aqueoussolutions. Chelating compounds comprising such esters may be added toaqueous reaction mixtures or mixed aqueous/organic reaction mixtures(i.e., for the radiolabeling and protein conjugation reactions) withrelatively little hydrolysis of the ester group. Thus, suchhydrolysis-resistant esters are particularly useful in reactions withproteins, since such reactions preferably are conducted under aqueousconditions to prevent denaturation of the proteins, which may occur inorganic solvents. Advantageously, the ester is sufficiently stable toallow preparation of the chelating compound ahead of time and storage,even under humid conditions, until needed, with the ester groupremaining substantially intact.

In addition, preferred esters are active esters. The term "active ester"is known to refer to esters which are highly reactive in nucleophilicsubstitution reactions. Preferred active esters for use in the presentinvention are highly reactive toward groups on polypeptides such thatthe ester-containing chelate compounds are bound to the polypeptidesthrough the reaction. These active esters- comprise leaving groups(i.e., the --OR' portion of an ester ##STR6## which are sufficientlyelectron-withdrawing to increase the susceptibility of the carbonyl##STR7## of the ester to attack by nucleophilic groups on the protein toform bonds. The kinetics of the reaction preferably are such that theester reacts quickly with nucleophilic groups on the polypeptide to formbonds. Thus, the esters are free (i.e., unreacted) ester groups,potentially susceptible to hydrolysis (especially if the reaction isconducted at a basic pH), for only a short time. Hydrolysis of theester, therefore, is further minimized, and a relatively high ratio ofthe desired aminolysis reaction to hydrolysis of the ester results.

Another consideration in choosing a suitable ester group is avoidance ofesters which would have decreased reactivity toward the polypeptide dueto steric hindrance. For example, increasing the size of the leavinggroup of the ester may cause steric hindrance of the reaction betweenthe ester and the polypeptide.

The leaving group also should not render the chelating compound orchelate derived therefrom (i.e., after radiolabeling) insoluble inwater. As described above, an important property of the chelating andchelate compounds of the invention is that they are sufficiently watersoluble to allow the compounds to be reacted with proteins in aqueous ormixed organic-aqueous solutions in which the organic solventconcentration is low enough to prevent damage to the protein (e.g.,denaturation). Examples of leaving groups which may render the chelatingcompound relatively insoluble in water include large nonpolar groupssuch as long chain hydrocarbons.

Common esters which find use are the o- and p-nitrophenyl,2-chloro-4-nitrophenyl, cyanomethyl, 2-mercaptopyridyl,hydroxybenztriazole, N-hydroxysuccinimide, trichlorophenyl,tetrafluorophenyl, 2-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl,o-nitro-p-sulfophenyl, N-hydroxyphthalimide, N,N-diethylamino,N-hydroxypyrrolidone, tetrafluorothiophenyl, and the like. For the mostpart, the esters will be of activated phenols, particularlynitro-activated phenols and cyclic compounds based on hydroxylamine. Asother hydroxylic compounds become available, these also may find use inthis invention.

Especially good results have been achieved by using a2,3,5,6-tetrafluorophenyl ester, which is an active ester having theabove-described properties of stability and high reactivity. As shown inthe examples below, chelate compounds of the present inventioncomprising the tetrafluorophenyl ester as the Y' group demonstratedrelatively efficient amide bond formation when reacted with compounds(including antibodies) containing free amines. Good results also havebeen achieved by using a thiophenyl ester as demonstrated in Example 18below.

The use of esters comprising nitro groups may be disadvantageous incertain circumstances. For example, the nitro group may be reduced bystannous ion which may be present when the stannous ion is added as apertechnetate or perrhenate reducing agent, as described below.

The polypeptide compounds may be varied widely, depending upon thenature of the use of the radionuclide. Thus, the polypeptides may be,among others, receptors, hormones, lymphokines, growth factors,substrates, particularly compounds binding to surface membranereceptors, where the complex may remain bound to the surface or becomeendocytosed. Among receptors are surface membrane receptors, antibodies(including monoclonal antibodies) enzymes, naturally occurringreceptors, lectins, and the like. Of particular interest areimmunoglobulins or their equivalent, which may involve Fab fragments,Fab' fragments, F(ab')₂, F_(v), T-cell receptors, etc.

Thus, "Y" may be a protein, a polypeptide or a fragment thereof. As usedherein, the term "polypeptide" includes polypeptides, proteins, orfragments thereof. These proteins and polypeptides may be modified aslong as the biological activity necessary for the intended diagnostic ortherapeutic application of the radiolabeled polypeptide is retained. Forexample, a modified antibody or fragment thereof may be used as long asbinding to the desired antigen still occurs. The amino acid sequence ofa protein may be varied (e.g., by known mutation techniques or deletionof portions thereof) as long as the desired biological activity (e.g.,binding of the protein to specific target cells, tissues or organs) isretained. Methods of modifying proteins also may include, among others,attachment of bifunctional linker compounds which react with both agroup on a protein and with the "Y" group on the chelating compounds,thereby binding the chelating compound to the protein through thelinking compound. The polypeptide may be purified from a natural sourceor may be synthetic (e.g., produced by recombinant DNA technology orchemical synthesis procedures).

Polypeptides which bind to the desired target site are said to be"specific for" the target site. For example, antibodies which bind to aparticular antigen are said to be specific for that antigen. It is to beunderstood that such polypeptides or antibodies are rarely 100% specificfor a target site, and a certain degree of cross-reactivity with othertissues is common, as discussed more fully below. An example of a targetsite is a cancer site. Many antigens associated with various types ofcancer cells have been identified, and monoclonal antibodies specificfor a number of these cancer cell-associated antigens also are known.Such antibodies are examples of the many polypeptides suitable for useas the "Y" or "Y'" component, which bind to a desired target site.

Proteins generally contain a variety of functional groups which mayreact with a Y or Y' group on the chelate compounds of the invention tobind the compounds to the protein. For example, when Y or Y' is theleaving group of an ester, the ester may react with hydroxy groups(e.g., on serine residues) or with sulfhydryl groups (e.g., on cysteineresidues, although the resulting bond may not be very stable). However,the active ester groups are believed to react preferentially with freeamino groups (generally those on lysine residues) in an aminolysisreaction. The resulting amide bond between the chelate compound and theprotein is relatively strong, stable and essentially irreversible underthe conditions which preserve the biological activity of the protein.Alternatively, when Y or Y' is an --NH² or --NHNH₂ group, an imine orhydrazine bond is formed between the chelating compound and the proteinthrough reaction with oxo groups on the protein (e.g., oxo groupsproduced on glycoproteins as described above).

The w,(w-x)-diamino aliphatic carboxylic acids, particularly alkanoicacids, generally will be of from 4 to 10, usually from 4 to 7 carbonatoms and are known compounds, or can be readily prepared inconventional ways or as described herein. For example, vicinaldibromides may be combined with aqueous ammonia under mild conditions.The amino groups may then be derivatized by reacting the hydrochloridesalt of the diamino ester (e.g., lower alkyl ester) with an α-haloacylchloride (e.g., chloroacetyl chloride) in an inert hydrocarbon solvent(e.g., toluene), followed by substitution of the chloro groups with amercapto group employing an appropriate derivative of hydrogen sulfide(e.g., sodium benzthiolate, sodium thioacetate, t-butyl mercaptan or thelike). The ester may now be hydrolyzed to the acid and the metal chelateformed or the thioether reacted with an activated sulfonyl chloridefollowed by treatment with thioglycolate. Alternatively α-alkylthiosubstituted acyl compounds may be used with carbodiimide for acylation,followed by cleavage of the thioether with formation of disulfide andreduction of the disulfide to mercapto, as described above.

An alternative approach, employed for the 4,5-diaminopentanoate, employsthe readily available glutamate. After forming the 5- carboxy ester, theamino group is protected and the acid group (1-carboxyl) preferentiallyreduced to the alcohol. The alcohol is transformed into an-activecleaving group (e.g., halide or pseudohalide), followed by displacementwith a nitrogen anion (e.g., azide), which serves as an intermediate tothe amino group. After catalytic reduction of the amino intermediate toamino and hydrolysis of the ester, the amino groups are acylated withS-protected α-mercaptoacyl groups. The protective groups may be removed,exchanged or otherwise modified (e.g., by introduction of watersolubilizing groups).

Various synthetic procedures may be employed for preparing the differentN₂ S₂ chelate rings. Carboxamides may be formed and reduced usingaluminum or borohydrides to form the amine. Amines may be alkylated withaliphatic halides. Ethylene or propylene diamines orcarboxyalkylalkylene diamines may be used to link thioglycolic acids.Other synthetic procedures may also be employed depending on the N₂ S₂ligand of interest.

The imidate may be employed by preparing the nitrile of the aminoprotected w,(w-x)-diaminoalkyl halide or pseudohalide by displacementwith nitrile, mercaptoacylation of the deprotected amino groups asdescribed previously and imidoester formation by conventionaltechniques, e.g., acidic (HCl) anhydrous alkanol.

The S-protective groups may be varied widely, being acyl groups, thiogroups or other compound which provides protection of the thio groupduring the subsequent manipulations and can be readily removed withoutdeleterious effect on the peptide conjugate.

The sulfur-protective groups also serve to stabilize the chelatingcompounds by preventing reaction of the sulfurs with groups which arepart of the chelating compound itself. For example, if protecting groupsT or T' were replaced with hydrogens, the sulfurs may displace an activeester group (Y or Y') from the chelating compound.

Illustrative groups include benzoyl, acetyl, m- or p-phthaloyl,thioglycolic, o-carboxythiophenol, ethylthiocarbonate,β-mercaptopropionic, tetrahydropyranyl, sulfonato, etc. Alternativelycyclic di- or polysulfides may be formed. Disulfides may be preparedusing sulfinyl halides, dinitrothiophenoxide substituted mercaptans,with mild oxidation in the presence of excess of the protective group,etc.

The protective groups may be removed in a variety of ways. Thioestersmay be hydrolyzed using aqueous ammonia, sodium alkoxide in alkanol, orany conventional technique. Disulfides may be cleaved withdithiothreitol, glutathione, β-mercaptoethylamine or other conventionalreagent. Cleavage of the disulfide may occur prior to or afterconjugation to the polypeptide.

In a preferred embodiment of the invention, the sulfur-protecting groupsT and T', when taken together with the two sulfur atoms to be protected,represent thioacetals or hemithioacetals. When such sulfur-protectinggroups are used, radiolabeling of the chelating compound with technetiumor rhenium may be accomplished efficiently under conditions oftemperature and pH which leave the ester group on the chelating compoundintact. The radiolabeling step may be accomplished in an exchangereaction under acidic pH conditions. When other types of protectinggroups are employed, the radiolabeling step generally is conducted at abasic pH and/or relatively high temperatures. Such conditions maydestroy the ester group. In addition, the reaction mechanisms may beother than an exchange reaction in other radiolabeling procedures.

The use of thioacetal or hemithioacetal S-protecting groups has theadvantage of simplifying the preparation of the radiolabeled chelatecompounds of the invention and the radiolabeled polypeptides preparedtherefrom. For example, a separate step for removal of thesulfur-protecting groups is not necessary. The protecting groups aredisplaced from the compound during the radiolabeling in what is believedto be metal-assisted acid cleavage; i.e., the protective groups aredisplaced in the presence of the metal radioisotope at an acidic pH, andthe radioisotope is bound by the chelating compound. In general, thehemithioacetal protective groups are somewhat more acid labile in theradiolabeling reaction than the thioacetal protective groups and,therefore, are generally preferred.

In addition, base-sensitive functional groups on the chelating compoundsurvive the radiolabeling step intact. This is especially advantageouswhen Y or Y' is an ester group. When radiolabeling is conducted at basicpH (especially at a pH above about 9 or 10), such ester groups aresubstantially hydrolyzed and must be generated (or regenerated) afterthe radiolabeling step. Generation of the ester group generally involvesa multistep procedure (e.g., by using a carbodiimide and a hydroxyliccompound, as described below and in Example 3). These extra steps, andthe need to remove carbodiimide and phenolic compounds (which may damagethe protein when it is added) from the reaction mixture, are avoidedwhen thioacetal and hemithioacetal protecting groups are used. Chelatingcompounds comprising esters thus are ready for conjugation to proteinsimmediately after radiolabeling without any esterification steps.

Thioacetals and hemithioacetals which may be used in the presentinvention include those groups which effectively maintain the sulfurs ina nonreactive state until the radiolabeling step, at which time theprotective groups are displaced in the presence of the metallicradioisotope under acidic conditions. When a thioacetal group is used, asingle protecting group protects both sulfurs shown in the formula forthe chelating compund. Thus, the two Ts in the formula are takentogether to represent a group which, together with the sulfur atoms tobe protected, defines a thioacetal group. Suitable thioacetals generallyhave the following formula in which the two sulfur atoms shown are thesulfur atoms of the chelating compound: ##STR8## wherein R¹ and R² arethe same or different and are selected from hydrogen; lower alkyl groups(preferably of from one to three carbon atoms, most preferably a methylgroup); or an aromatic (phenyl) ring with an electron donating group(e.g., a methoxy, ethoxy, or hydroxy group, with methoxy beingpreferred) bonded directly to the ring, preferably in the para position.When either R¹ or R² comprises a phenyl ring, the other preferably ishydrogen so that the desired degree of water solubility is retained.Alkyl groups comprising longer carbon chains generally would decreasethe water solubility of the chelating compound. Examples of suitablethioacetals include, but are not limited to, p-anisylidine: ##STR9## andacetonyl: ##STR10##

When hemithioacetal protective groups are used, each T or T', when takentogether with a sulfur atom to be protected, defines a hemithioacetalgroup.

Suitable hemithioacetals include, but are not limited to, those havingthe following formulae, wherein the sulfur atom is a sulfur atom of thechelating compound, and a separate protecting group is attached to eachof the two sulfur atoms on the chelating compound:

--S--CH₂ --O--CH₂ --CH(CH₃)₂ --SCH₂ NHCOCH₃ --SCH₂ OCH₃

--S--CH₂ O--(CH₂)₂ --OCH₃

Preferred hemithioacetals generally are of the following formula,wherein the sulfur atom is a sulfur atom of the chelating compound and aseparate protecting group (T or T') is attached to each of the twosulfur atoms on the chelating compound: ##STR11## wherein R³ is a loweralkyl group, preferably of from two to five carbon atoms, and R⁴ is alower alkyl group, preferably of from one to three carbon atoms.Alternatively, R₃ and R⁴ may be taken together with the carbon atom andthe oxygen atom shown in the formula to define a nonaromatic ring,preferably comprising from three to seven carbon atoms in addition tothe carbon and oxygen atoms shown in the formula. R⁵ represents hydrogenor a lower alkyl group wherein the alkyl group preferably is of from oneto three carbon atoms. Examples of such preferred hemithioacetalsinclude, but are not limited to: ##STR12##

In general, the above-described thioacetals and hemithioacetals shouldnot comprise long hydrocarbon chains. Such chains would diminish thedesired water solubility of the chelating compounds of the invention andmay decrease the ease of synthesis thereof.

Depending upon the particular metal, various conditions and techniqueswill be employed for preparing the metal chelate. To prepare thetechnetium chelate, the chelating compound as carboxylate or activeester may be combined with a pertechnetate solution in the presence of areducing agent (e.g., stannous ion or dithionite under conventionalconditions), whereby the technetium chelate is formed as a stable salt.The rhenium chelate may be formed by reducing perrhenate with stannousion in the presence of citrate and the N₂ S₂ ligand. Yields generallyare 50% or greater after 1 hour at 50° C. Chelates of ²¹² Pb, ²¹² Bi and¹⁰⁹ Pd may be prepared by combining the appropriate salt of theradionuclide with the chelating compound and incubating the reactionmixture at room temperature or at higher temperatures. It is notnecessary to treat the lead, bismuth, palladium, and copper isotopeswith a reducing agent prior to chelation, as such isotopes are alreadyin an oxidation state suitable for chelation.

The chelating agent may be already esterified or esterified inaccordance with conventional ways. If already esterified, a labilecomplex such as Tc-99m gluconate or glucoheptonate may be prepared whichwill allow exchange to the N₂ S₂ active ester ligand, forming a complexsuitable for protein conjugation. Alternatively, the carboxylic acid maybe activated by employing a water soluble carbodiimide (e.g., EDCI) inan aqueous medium in the presence of at least a stoichiometric amount,preferably an excess, of the hydroxylic compound. A suitably bufferedaqueous medium may be employed. Excess carbodiimide can be converted tourea by adding acetate. The aqueous medium may then be used directlywithout further purification for conjugation to the polypeptide.Desirably, the polypeptide will be added to the ester containing aqueousmedium at a convenient concentration at a mildly alkaline pH, generallyin excess of about 7.5 and less than about 9 and the reaction allowed toproceed for a sufficient time for virtually all of the active ester toeither react with the polypeptide or be substantially completelyhydrolyzed. Usually, the time will be less than about 6 hours and morethan about 30 minutes, with temperatures ranging from about 0° to 50°C., usually not exceeding about 40° C. In general, the reaction time maybe decreased when more highly active esters are used. For example, whena chelate comprising a tetrafluorophenyl ester is used, the reaction ofthe chelate with a protein may be substantially complete in 20 minutes,as described below (e.g., in Example 15). The particular conditions willbe selected in accordance with the particular active ester, the pH, theactivity of the polypeptide, and the like.

If desired, the number of free amino groups (i.e., those available forreaction with the chelate) on a particular polypeptide or fragmentthereof may be estimated by known methods. See, for example, Snyder andSobocinski (Analytical Biochemistry, 64:284-288 [1975]) and Habeeb(Analytical Biochemistry, 14:328-336 [1966]). Basically, an assay usingtrinitrobenzene sulfonic acid (TNBS) with glycine as the standard may beperformed. Either the standard or a sample of the polypeptide isdissolved in 0.1 M sodium borate, pH 9.2. TNBS is added to a finalconcentration of 0.75 mM, and the solution is allowed to stand for 30minutes at room temperature. The absorbance at 420nm is then read. Theassay is linear over the range 1×10⁻⁸ to 2×10⁻⁷ moles of free aminegroups. The mount of antibody added to the conjugation reaction may beadjusted to give the desired stoichiometry (e.g., a 1:1 ratio of chelateto free amine groups).

It is also feasible but less preferable to conjugate the chelating agent(N₂ S₂) to the polypeptide in the absence of the metal ion. The Y or Y'group would be linked to the polypeptide to form a stable covalent link(e.g., an amide linkage), followed by the addition of the metal in areduced, chelated, exchangeable form. As chelates, α- or β-dioxocompounds are useful. Conveniently, the metal ion could be added as aweakly chelated ion or in the presence of a weakly chelating group, suchas a uronate (e.g. gluconate).

A disadvantage of conjugating the chelating compound to a polypeptidebefore the radiolabeling step is that the radioisotope may bind to othersites on the polypeptide in addition to binding to the chelatingcompound. The non-specifically bound (i.e., unchelated) radioisotope maybe only weakly attached and may later detach from the antibody andinterfere with the diagnostic or therapeutic technique for which theradiolabeled polypeptide is to be used.

The subject chelate polypeptide conjugates (i.e., radiolabeledpolypeptides having a chelate compound of the invention bound thereto)will be administered to the mammalian host, normally by injection,intravenously, intra-arterially, peritoneally, intratumorally, or thelike, depending upon the particular site at which the radionuclide isdesired. Generally, from about 0.1 to 2 mL will be injected into a hostfor diagnostic purposes, depending upon the size of the host, with about0.001 to 50 uCi/kg of host. For human hosts the dosage will usually beabout 10-50 mCi/70 kg host, more usually about 25-35 mCi/70 kg host.When the chelate polypeptide conjugates are to be injected into thebloodstream of a human, the total volume injected may be larger, e.g.,20 to 30 mls administered by intravenous infusion, as described inExample 15. For lower mammals (e.g., mice), about 1 to 50 uCi isadministered for biodistribution studies, while up to or greater than500 uCi is administered for imaging studies. After administration of theradionuclide, depending upon its purpose, the host may be treated invarious ways for detection or therapy.

The diagnostic uses of the chelate-polypeptide conjugates of theinvention thus provide a method for detecting the presence or absence ofa particular target site within a human or mammalian host. In general,such a conjugate (e.g., a compound as shown in the formula above inwhich M is a ^(99m) Tc radionuclide and Y' is a polypeptide which bindsto said target site) is administered to the host, and thebiodistribution of the ^(99m) Tc is detected after waiting apredetermined length of time to allow accumulation of the compound atthe target site. The diagnostic procedures may vary according to thepolypeptide component of the conjugate and other factors. One suchprocedure is described in more detail in Example 17 below.

Technetium-99m (^(99m) Tc) has a physical half-life of 6 hours. Wholeimmunoglobulins have a biological half-life in serum of approximately 24hours (wide range), and thus the clearance of ^(99m) Tc-labeled antibodyfrom the circulation is slow compared to the physical half-life of^(99m) Tc. A ^(99m) Tc-labeled F(ab')₂ fragment has a shortercirculation time (T1/2 9-20 hours) than whole immunoglobulin, which ismore compatible with tumor localization and background clearance for the^(99m) Tc-labeled antibody fragment to provide sufficienttumor:background ratios to image lesions successfully. Smaller fragmentssuch as Fab', Fab and Fv have shorter circulation times (T1/2 less than180 minutes) that are more compatible with the physical T1/2 of ^(99m)Tc and are thus preferred for imaging applications. Choice of molecularspecies of antibody for imaging with other radionuclides will similarlydepend on the relationship of the physical half-life of the radionuclideand the circulation time of the molecular species of antibody. ⁶⁷ Cu,with a physical half-life of 58.6 hours, can be used with whole, F(ab')₂or smaller fragments.

Choice of molecular species of antibody for therapy applications ofradionuclides is more complex. In addition to physical and biologichalf-lives, residence time of the labeled antibody in the tumor, energyof the emission and contribution of total body to specific organ doseare critical issues that dictate the optimal size of antibody orfragment. With monoclonal antibodies, the particular antibody will alsobe a factor influencing the choice.

Rhenium-188 (¹⁸⁸ Re) has a 17-hour physical half-life, for which F(ab')₂and Fab antibody fragments have suitable serum half-lives for tumorlocalization and background clearance. The ¹⁸⁸ Re-labeled Fab would beexpected to cause less toxicity to the bone marrow, but it will usuallyhave a shorter residence time in tumor due to the lower affinity ofunivalent compared to bivalent fragments. A ¹⁸⁸ Re-labeled Fab fragmentwith a suitably high affinity to maximize tumor residence of thedelivered counts is especially useful.

Rhenium-186 (¹⁸⁶ Re) has a 3.67 day physical half-life. It can be usedwith whole antibody or F(ab')₂ or smaller fragments thereof. Because thebeta energies are decreased compared to ¹⁸⁸ Re, the labeled antibodywill need to have a longer residence time in the tumor.

¹⁰⁹ Pd has a half life of 14 hours. Antibody fragments, as opposed towhole antibodies, are expected to generally be most suitable forradiolabeling in accordance with the invention.

²¹² Pb has a physical half-life of 10.8 hours. Fab', Fab or Fv fragmentsradiolabeled with ²¹² Pb would provide the greatest tumor uptake andbackground clearance in that period. ²¹² Pb decays to ²¹² Bi which hasan alpha emission with a physical half-life of 60 minutes. ²¹² Bi itselfis not a feasible label unless compartmental administration (e.g.,intraperitoneal) is used. ²¹² Pb will transmute to ²¹² Bi in situ, andit is necessary to use a ligand that can withstand the recoil fromB-decay.

⁶⁷ Cu has a physical half-life of 2.44 days. In general, wholeantibodies or F(ab')₂ fragments thereof are most suitable forradiolabeling with this isotope for therapeutic use.

Delivery of the radiolabeled polypeptide may occur intravenously or byintraperitoneal, intralymphatic, intrathecal, or other intracavitaryroutes. Advantageously, an unlabeled (non-radiolabeled) antibodyreactive with the same epitope as a radiolabeled antibody of theinvention is administered prior to administration of the radiolabeledantibody, as described in the co-pending U.S. Patent Application havingSer. No. 917,176. The non-radiolabeled antibody functions as an"unlabeled specific blocker" to decrease binding of thelater-administered radiolabeled antibody to cross-reactive sites whichmay be present on non-target tissue. Blocking of such cross-reactivesites is important because antibodies generally have somecross-reactivity with tissues other than a particular target tissue. Inthe case of antibodies directed again tumor-specific antigens, forexample, virtually all such antibodies have some cross-reactivity withnormal (i.e., non-tumor) tissues with the exception of anti-idiotypes toB-cell lymphoma.

Antibodies to the 250 Kd glycoprotein/proteoglycan melanoma-associatedantigen have been labeled with ^(99m) Tc as disclosed in Examples 14 and15 below. It has been discovered, for example, that prior injection ofunlabeled anti-250 Kd antibody as an unlabeled or cold specific blockerdecreases uptake of labeled antibody in spleen and bone marrow (seeco-pending U.S. patent application having Ser. No. 917,176), and thusimproves tumor localization.

The unlabeled (cold) specific blocker polypeptide advantageously isadministered from about 5 minutes to about 48 hours, most preferablyfrom about 5 minutes to about 30 minutes, prior to administration of theradiolabeled polypeptide. The length of time may vary according to suchfactors as the nature of the antibody and the relative accessibility oftarget sites versus cross-reactive binding sites. The unlabeled specificblocker and the radiolabeled antibody may be the same (except for theradiolabeling) or different, as long as both recognize the same epitope.In one embodiment of the invention, the unlabeled specific blocker is abivalent form of an antibody (e.g., a whole antibody or a F[ab']₂fragment thereof) and the radiolabeled polypeptide is a monovalentfragment of the same antibody (e.g., a F[ab]', F[ab], or Fv fragment).Use of a bivalent form of an antibody as the cold specific blocker and amonovalent form for the radiolabeled antibody has the advantage ofminimizing displacement of the blocker from cross-reactive sites by thelater administered radiolabeled antibody due to the greater affinity ofthe bivalent form. The unlabeled specific blocker polypeptide isadministered in an amount effective in binding with (blocking) at leasta portion of the cross-reactive binding sites in a patient. Thus,binding of a radiolabeled polypeptide to cross-reactive binding sitesmay be reduced, thereby improving diagnostic imaging of target sites,and in general, reducing somewhat the amount of radiolabeled antibody tobe administered. The amount may vary according to such factors as thesize of the patient and the nature of the polypeptide. In general, about5 mg or more of the unlabeled specific blocker is administered to ahuman.

Advantageously, a second antibody, termed an "irrelevant" antibody, alsois administered to a patient prior to administration of the radiolabeledpolypeptide. The irrelevant antibody is an antibody which does not bindto sites within the patient by a specific (e.g., antigen-binding)mechanism but which may bind to target and non-target sites throughnon-specific mechanisms (e.g., adsorption or binding of the Fc portionof the irrelevant antibody to Fc receptors on cells in thereticuloendotheial system). The irrelevant antibody blocks certainnon-target sites in a patient and thus decreases non-specific binding ofthe radiolabeled polypeptide to these non-target sites, as described incopending U.S. patent application Ser. No. 917,176. Diagnostic imagingof target sites thus may be improved, and the amount of radiolabeledantibody to be administered may be somewhat reduced. For example, prioradministration of an irrelevant antibody which is not specific for anyhuman tissues, as far as is known, effectively reduced the non- specificuptake of whole and F(ab')₂ radiolabeled antibody into liver and spleenin human patients.

The irrelevant antibody advantageously is administered from 5 minutes to48 hours, most preferably from 15 minutes to one hour, prior toadministration of the radiolabeled polypeptide. The length of time mayvary according to such factors as the nature of the antibody. Manysuitable antibodies which may be used as the irrelevant antibody areknown. For example, there are many known antibodies which are notspecific for any human tissues, which may be used as the irrelevantantibody. In one embodiment of the invention, a murine monoclonalantibody to a B-cell lymphoma idiotype (i.e., specific for the lymphomacells only of one individual human) is administered as the irrelevantpolypeptide. In one embodiment of the invention, the irrelevantpolypeptide is a whole antibody or a F(ab)'₂ fragment thereof. Theirrelevant polypeptide is administered in an amount effective inblocking at least a portion of the sites at which non-specific binding(i.e., binding through non-specifc mechanisms) of the radiolabeledpolypeptide occurs in the absence of the irrelevant polypeptide. Theamount may vary according to such factors as the nature of thepolypeptides and the size of the patient. In general, about 15 mg ormore (preferably less than 200 mg) of the irrelevant antibody isadministered.

In another embodiment of the invention, the above-described chelatingcompounds may be included in a kit for producing a chelate-polypeptideconjugate of the invention for radiopharmaceutical use. Preferably, aspecific polypeptide to be radiolabeled (as described above) also isincluded in the kits. Reagents useful in reactions to radiolabel thechelating compound with a radionuclide and to conjugate the resultingchelate compound to the polypeptide also may be included. Such kits alsomay comprise a means for purifying the radiolabeled polypeptide from thereaction mixture, as well as specific instructions for producing theradiolabeled polypeptide using the kit components. Such kits generallywill be used in hospitals, clinics or other medical facilities. Sincesuch facilities generally have ready access on a daily basis toradionuclides such as isotopes of technetium, and since isotopes ofrhenium, lead, bismuth, palladium, and copper may be prepared asdescribed above, inclusion of the radionuclide in the kit is optional.Exclusion of the radionuclide permits storage of the kit, whereas kitscontaining the radionuclide (either as a separate component or as theradiolabeled chelate compound) would have to be used within a narrowtime frame (depending on the half-life of the particular isotope);otherwise, radioactive decay of the radioisotope would diminish theeffectiveness of the diagnostic or therapeutic technique for which theradiolabeled protein is used. For ¹⁸⁶ Re, on-site radiolabeling wouldavoid radiolytic degradation of the labeled antibody due to the betaparticle emission.

The kits may be diagnostic or therapeutic kits, depending on whichradioisotope is used for labeling the chelating agent. When theradionuclide is to be reduced to a lower oxidation state (e.g.,technetium and rhenium, as discussed above), the kits may additionallycomprise a reducing agent effective in reducing a particular metalradionuclide, to be chelated by the chelating compound, to an oxidationstate at which an exchange complex of the radionuclide may be formed,and a complexing agent with which said reduced radionuclide will formsaid exchange complex. The kit components and instructions will besomewhat different when the chelating agent is to be radiolabeled with atechnetium isotope (i.e., a diagnostic kit) than when the chelatingagent is to be radiolabeled with a rhenium, lead, bismuth, palladium, orcopper isotope (i.e., a therapeutic kit). The different components andprocedures are discussed in more detail below.

Since the chelating compounds preferably are radiolabeled with aradionuclide prior to conjugation to a protein, a kit preferablyincludes a chelating compound comprising sulfur-protecting groups and apolypeptide in separate containers instead of a single containercontaining a chelating compound already conjugated to the protein. Theterm "separate containers" as used herein is meant to include not onlyseparate, individual containers (e.g., vials) but also physicallyseparate compartments within the same container. Thus, the radiolabeledchelate is prepared by the procedures described below, then conjugatedto the polypeptide.

As discussed above, the procedures for preparing a radiolabeled proteinaccording to the present invention may be simplified by using theabove-described hemiacetals and hemithioacetals as sulfur-protectinggroups. Thus, the chelating compound included in a kit preferablycomprises thioacetal or hemithioacetal S-protecting groups. Preferredchelating compounds also comprise the above-described active esters,which remain intact and are reactive with the polypeptide afterradiolabeling in an exchange reaction under acidic conditions, asexplained above.

In accordance with one embodiment of the invention, a diagnostic kitcomprises the following reagents (in separate containers unlessotherwise noted), presented in the general order of use.

1. A reducing agent effective in reducing pertechnetate (^(99m) Tc)₄ --which is in the +7 oxidation level) to a lower oxidation state at aneutral to acidic pH so that a technetium exchange complex can beformed. Many suitable reducing agents are known, including but notlimited to stannous ion, (e.g., in the form of stannous salts such asstannous chloride or stannous fluoride), metallic tin, formamidinesulfinic acid, ferric chloride, ferrous sulfate, ferrous ascorbate, andalkali salts of borohydride. Preferred reducing agents are stannoussalts.

2 A complexing agent with which the reduced ^(99m) Tc will form anexchange complex, thus protecting the ^(99m) Tc from hydrolysis. Inorder to achieve efficient transfer or exchange of the ^(99m) Tc fromthis complex to the chelating compound, the complexing agentadvantageously binds the radionuclide more weakly than the chelatingagent will. Complexing agents which may be used include, but are notlimited to, gluconic acid, glucoheptonic acid, methylene diphosphonate,glyceric acid, glycolic acid, mannitol, oxalic acid, malonic acid,succinic acid, bicine, N,N'-bis(2-hydroxyethyl) ethylene diamine, citricacid, ascorbic acid and gentisic acid. Good results are obtained usinggluconic acid or glucoheptonic acid as the Tc-complexing agent (or"exchange agent" in these cases), as they efficiently transfer the^(99m) Tc to the N₂ S₂ chelating agent

3. A chelating compound of the invention suitable for binding to thepolypeptide component of the kit, as described above.

4. A protein, polypeptide or fragment thereof specific for the desiredtarget organ, tissue, antigen or other target site within a mammalianbody, as discussed above.

5. Means for purifying the desired chelate-polypeptide conjugate fromthe reaction mixture. Any suitable known protein purification techniquemay be used which effectively separates the desired radiolabeled proteinconjugate from other compounds in the reaction mixture. The purificationstep may, for example, separate the desired conjugate from impuritiesdue to differences in size or in electrical charge. One suitablepurification method involves column chromatography, using, for example,an anion exchange column or a gel permeation column. Good results havebeen achieved by column chromatography using an anion exchange column,e.g., a quaternary amino ethyl Sephadex (QAE-Sephadex) column or adiethyl aminoethyl Sephadex (DEAE-Sephadex) column. Since virtually allthe impurities to be removed (e.g., Tc-gluconate, sodium pertechnetate,technetium dioxide and the hydrolyzed--i.e., carboxylate--form of thechelate) are negatively charged, they are substantially retained on thepositively charged column. Purification thus may be accomplished by thisone-step column procedure.

6. Additional reagents for use in the radiolabeling and proteinconjugation reaction mixtures (e.g., the buffers, alcohols, acidifyingsolutions, and other such reagents, as described below) are generallyavailable in medical facilities and thus are optional components of thekit. However, these reagents preferably are included in the kit toensure that reagents of sufficient purity and sterility are used, sincethe resulting protein conjugates are to be administered to mammals,including humans, for medical purposes.

7. Optionally, a container of a polypeptide to be administered innon-radiolabeled form to a human or mammal is included in the kit. Thispolypeptide is reactive with essentially the same target site as thepolypeptide to be radiolabeled and reduces binding of the radiolabeledpolypeptide to cross-reactive binding sites on non-target tissues. Thetwo polypeptides may be the same, or the polypeptide to be radiolabeledmay, for example, be a fragment of the polypeptide which is to beadministered in non-radiolabeled form. The latter polypeptide isadministered as an unlabeled specific blocker (prior to administrationof the radiolabeled polypeptide) in an amount effective in improvingdiagnostic imaging of the desired target sites (e.g., tumors) asdescribed above.

8. Optionally, the kit also comprises a container of a polypeptide whichdoes not bind through specific mechanisms to sites within the human ormammal to which the radiolabeled polypeptide is to be administered. Thispolypeptide is administered as an "irrelevant" polypeptide (prior toadministration of the radiolabeled polypeptide) in an amount effectivein decreasing nonspecific uptake of certain radiolabeled polypeptides,as described above.

In one embodiment of the invention, a radiolabeled polypeptide may beproduced using such a kit according to the following general procedure.The procedure is conducted under sterile conditions. In this particularembodiment of the invention, the kit comprises reagents in amountssuitable for preparation of an amount of radiolabeled polypeptidesuitable for injection into one human for diagnostic purposes.

An aqueous solution comprising a reducing agent and a complexing agentis prepared. Good results are achieved by combining stannous chloridedihydrate (comprising the stannous ion reducing agent) and sodiumgluconate (a complexing agent) to form a stannous gluconate complex.This stannous gluconate complex may be provided in a single container inthe kit. In one embodiment of the invention, the stannous gluconatecomplex is provided in the kit in dry solid form. Optionally, one ormore stabilizer compounds may be added to the stannous gluconatecomplex. Many such stabilizer compounds are known and are discussed inconnection with the therapeutic kits below. For example, gentisic acidmay be added to a container of the stannous gluconate complex tostabilize (minimize oxidation of) the stannous ion reducing agent, andthe resulting mixture may be provided in the kit in dry solid form or asa lyophilized preparation. A filler compound advantageously is addedprior to lyophilization, as described for the therapeutic kit below. Forexample, lactose may be added as a filler compound in an amounteffective in facilitating lyophilization. The amounts of stannouschloride and sodium gluconate preferably are not so large as to haveadverse effects on the desired reactions and product. For example,excessively large amounts of non-reacted (free) stannous chloridedihydrate may harm the polypeptide added in a later step, e.g., byadversely affecting the immunoreactivity of an antibody. An excessivelylarge amount of free sodium gluconate may slow the transchelation stepand require addition of excessive amounts of buffer necessary to raiseth pH in subsequent steps, and the reaction mixtures would thenbeundesirably dilute. An acceptable ratio of stannous chloride dihydrateto sodium gluconate (by weight) is from about 1:10 to about 1:100,preferably from about 1:25 to about 1:70, most preferably about 1:41.6.

The amount of ^(99m) Tc added may vary. When the diagnostic kit isdesigned for preparation of a radiolabeled protein to be injected into asingle human patient, the amount of pertechnetate to be added to thefollowing reaction mixture may be from about 50 to about 200 mCi,preferably from about 75 to about 100 mCi of the radioisotope. Greateramounts may interfere with the reaction and produce low yields, as wellas being an excessive amount of radioactivity for administration to asingle patient, as described in the examples below. When about 75 to 100mCi of Tc0₄ ⁻⁻ are to be added, the stannous gluconate complexpreferably comprises (i.e., is formed from) about 3 to about 10 mg ofsodium gluconate and about 0.075 to about 0.250 mg of stannous chloridedihydrate; preferably from about 4 to about 6 mg of sodium gluconate andabout 0.075 to about 0.125 mg of stannous chloride dihydrate.

Sodium pertechnetate is combined with the reducing agent and complexingagent. When the sodium pertechnetate is added to stannous gluconate, theradioisotope is effectively reduced to a lower oxidation state andcomplexed with gluconate to form an exchange complex. The stannousgluconate and pertechnetate may be combined in various ways. In oneembodiment of the invention, sterile water is added to a vial containinga stannous gluconate preparation in dry solid form. A portion of theresulting solution is combined with about 0.75 mL sodium pertechnetate(about 75 to 100 uCi). In another embodiment of the invention, sodiumpertechnetate (about 1 mL) is added directly to a lyophilizedpreparation comprising stannous gluconate, gentisic acid as astabilizer, and lactose as a filler compound. In either case (both ofwhich are described more fully in example 15 below), the reactionmixture is incubated at about 25° C. to about 50° C., preferably atabout 25° C. to about 37° C. for a minimum of 10 minutes. Incubation for10 minutes generally gives sufficient yields of the desired technetiumexchange complex (e.g., technetium gluconate) while minimizing theformation of insoluble technetium dioxide, which may increase withincreased incubation time.

A chelating compound of the invention, comprising a thioacetal orhemithioacetal S-protecting group, as described above, is added to anorganic solvent effective in dissolving the chelating compound andsuitable for the exchange reaction that follows. Suitable solventsshould be nontoxic in mammals and inert toward the reactants in thereaction mixture. Organic solvents which may be used includeacetonitrile, ethyl acetate, and methylethyl ketone. When theradiolabeled protein is to be injected into humans, however, suitableorganic solvents include, but are not limited to, alcohols such asethanol, butanol, t-butyl alcohol and propanol, and polar aproticsolvents such as DMSO and dimethylformamide. The choice of solvent mayvary according to the particular chelating agent included in the kit.For example, when the chelating compound comprises a tetrafluorophenylester group, ethanol will react with the ester in a transesterificationreaction, producing ethyl ester as a by-product , which is much lessreactive toward free amine groups on proteins. A preferred organicsolvent is isopropyl alcohol. The concentration of the organic solventin the following Tc-labeling exchange reaction mixture should be betweenabout 10% and about 30%, preferably between about 15% and about 25%.

The solution comprising the chelating agent in the organic solvent isthen acidified to a pH of about 2.0 to about 5.0, preferably 2.8 to 3.3.At these acidic pH conditions, the formation of insoluble TcO₂ will beminimized, and, as explained above, hemithioacetal and thioacetalsulfur-protecting groups will be displaced by a metal-assisted acidcleavage during the technetium labeling exchange reaction to form thecorresponding technetium chelate compound. Also, hydrolysis of estergroups on the chelating compound is minimized under acidic conditionswhen compared to basic conditions. Suitable acids are added in amountssufficient to displace the sulfur-protective groups in the presence ofthe metal radionuclide (i.e., in amounts sufficient to adjust thereaction mixture to the above-described pH values range). Suitable acidsinclude, but are not limited to, phosphoric acid, sulfuric acid, nitricacid, glacial acetic acid, hydrochloric acid and combinations thereof.Also included are solutions comprising such acids and buffers (e.g.,acetate and phosphate buffers). Good results have been achieved using asolution comprising glacial acetic acid and 0.2 N HCl at a ratio of2:14.

The acidified chelating compound solution is combined with thepreviously prepared technetium exchange complex solution, to form thecorresponding chelate compound, such that about 100 ug to about 150 ug,preferably about 135 ug of chelating compound is combined with theTc-gluconate complex prepared from the 75 to about 100 mCi of technetiumas described above. The reaction mixture is heated to between about 50°C. and 100° C. for from about 5 minutes to about 45 minutes. Goodresults have been achieved by heating at about 75° C. for about 15±2minutes. Heating the reaction mixture accelerates the exchange reactionto form the N₂ S₂ chelate. Upon completion of the reaction, the mixtureis transferred immediately to a 0° C. ice bath for a minimum of 2minutes to stop the reaction quickly and minimize hydrolysis of theester group.

An aqueous solution comprising a buffer then is added to the reactionmixture to reduce the concentration of the organic solvent (e.g.,isopropanol) and to raise the pH before adding the polypeptide to beradiolabeled. Suitable buffers include nontoxic buffers which are inerttoward the reactants, such as, but not limited to, sodium phosphatebuffer and sodium bicarbonate buffer, preferably at a concentration ofabout 1.0 M and a pH of about 10. Buffers such as TRIS are not suitablebecause the free amine groups of TRIS are reactive with the ester groupon the chelate compound. Sufficient buffer is added to reduce theorganic solvent concentration to from about 10% to about 15%, preferablyfrom about 7.5% to about 12.5%, and to raise the pH of the Tc-chelatesolution to about 5.5. If the pH were raised to higher levels (e.g., pH9 or above) before addition of the polypeptide, the polypeptide wouldnot be available to react with the ester, which would remain as a freeester group subject to hydrolysis at the higher pH. A buffer, preferablythe same buffer, is added to a solution of the desired polypeptide. Thepolypeptide may be provided in the kit in any form in which the desiredbiological activity is preserved. The polypeptide may, for example, beprovided in a buffered solution having a biologically acceptable pH,i.e., a pH at which the polypeptide may be stored in the kit withoutsignificant loss of biological activity. In one embodiment of theinvention, the polypeptide is provided in the kit in phosphate bufferedsaline (PBS) at a pH of about 7.0 to about 7.4. The buffered polypeptidesolution is added to the buffered Tc-chelate prepared above. Sufficientbuffer is added to the protein solution so that the final pH of theprotein conjugation reaction mixture is from about 9 to about 11. Theconcentration of the protein in the conjugation reaction mixture shouldbe at least about 1 mg/mL to achieve adequate yields of radiolabeledprotein. Increasing the final protein concentration above about 8 mg/mLgenerally does not increase the yield significantly. Preferably, thefinal protein concentration is about 5 mg/mL. The reaction is incubatedat 0° C. to 37° C. for about 10 minutes to about 35 minutes, preferablyat about 20° C. to 25° C. for about 20 minutes. The pH and temperatureof the reaction mixture should be kept within physiologically acceptablelimits to prevent loss of biological activity of the protein. Theresulting radiolabeled protein is purified by known methods, asdescribed above. The method chosen may vary according to such factors asthe size of the protein or protein fragment.

In accordance with another embodiment of the invention, a therapeutickit comprises the following reagents.

1. A reducing agent effective in reducing ReO₄ --, which is in the +7oxidation level, to a lower oxidation state at a neutral to acidic pH sothat a rhenium exchange complex can be formed. Many suitable reducingagents are known, including but not limited to stannous ion (e.g., inthe form of stannous salt such as stannous chloride or stannousfluoride), metallic tin, formamidine sulfinic acid, ferric chloride,ferrous sulfate, ferrous ascorbate, and alkali salts of borohydride.Preferred reducing agents are stannous salts.

2. A complexing agent with which the reduced Re will form an exchangecomplex, thus protecting the Re from hydrolysis. In order to achieveefficient transfer or exchange of the Re from this complex to the N₂ S₂chelating compound, the complexing agent advantageously binds theradionuclide more weakly than the chelating agent will. Complexingagents which may be used include, but are not limited to, methylenediphosphonate, glyceric acid, glycolic acid, mannitol, oxalic acid,malonic acid, succinic acid, bicine, N,N'-bis(2-hydroxyethyl) ethylenediamine, citric acid, ascorbic acid, gentisic acid, tartric acid, andgluconic acid. Good results are obtained using citric acid as theRe-complexing agent (or "exchange agent" in these cases).

3. A chelating compound of the invention suitable for binding to thepolypeptide component of the kit, as described above.

4. A protein, polypeptide or fragment thereof specific for the desiredtarget organ, tissue, antigen or other site within a mammalian body, asdiscussed above.

5. Means for purifying the desired chelate-polypeptide conjugate fromthe reaction mixture. Any suitable known protein purification techniquemay be used which effectively separates the desired radiolabeled proteinconjugate from other compounds in the reaction mixture. The purificationstep may, for example, separate the desired conjugate from impuritiesdue to differences in size or in electrical charge. One suitablepurification method involves column chromatography. Good results havebeen achieved by column chromatography using a gel permeation column oran anion exchange column (e.g., a QAE-Sephadex or DEAE-Sephadex column).Since virtually all the impurities to be removed (e.g., Re-citrate,perrhenate and the hydrolyzed [i.e., carboxylate] form of the chelate)are negatively charged, they are substantially retained on thepositively charged column. Purification thus may be accomplished by thisone-step column procedure.

6. Optionally, the kit may contain another column for purification ofthe chelate after the radiolabeling step. Any suitable reverse-phasecolumn may be used, such as a C-18 or C-8 Baker column.

7. Additional reagents for use in the radiolabeling and conjugationreaction mixtures (e.g., the buffers, alcohols, acid solutions, etc., asdescribed below) are generally available in medical facilities and thusare optional components of the kit. However, these reagents preferablyare included in the kit to ensure that reagents of sufficient purity andsterility are used, since the resulting protein conjugates are to beadministered to mammals, including humans, for medical purposes.

8. The kit also may include a container of an antibody to beadministered as an unlabeled specific blocker, as well as a container ofan appropriate antibody to be administered as an irrelevant antibody.The unlabeled specific blocker and irrelevant antibodies areadministered as described above to improve localization of theradiolabeled polypeptide at the desired target site.

In one embodiment of the invention, a polypeptide radiolabeled witheither ¹⁸⁸ Re or ¹⁸⁶ Re may be prepared using such a kit, according tothe following general procedure. The procedure is conducted understerile conditions.

Perrhenate (the ReO₄ ⁻⁻ form of the ¹⁸⁶ Re or ¹⁸⁸ Re isotope) is reactedwith a reducing agent and a complexing agent. Good results are achievedby combining citric acid (a complexing agent) with stannous chloride (areducing agent) in a single container (in which a stannous citratecomplex is believed to form) and adding the perrhenate thereto.

The amounts of stannous chloride and citric acid added should not be solarge as to have adverse affects on the desired reactions. For example,excessively large amounts of non-reacted (free) stannous chloride mayharm the polypeptide added in a later step (e.g., by adversely affectingthe immunoreactivity of an antibody). An excessively large amount offree citric acid may lower the pH to a level which makes addition oflarge quantities of buffer necessary to raise the pH in subsequentsteps, and the reaction mixtures would be undesirably dilute. Anacceptable ratio of stannous chloride to citric acid (by weight)generally is from about 1:10 to about 1:5500, preferably from about 1:20to about 1:200, most preferably about 1:100.

One or more stabilizer compoundsmay be added to the stannous citratecomplex. Many such stabilizer compounds are known. See, for example,U.S. Pat. Nos. 4,440,738 and No. 4,510,125. Advantageously, gentisicacid is added to the stannous citrate to stabilize (e.g., to preventoxidation of) the stannous ion. The stabilizer is added to a solutioncomprising the stannous chloride reducing agent (and the complexingagent) in an amount effective in stabilizing the stannous ion such thatthe shelf life (stability) of the stannous ion is increased. Thesolution may be lyophilized and provided in the kit as a lyophilizedpowder.

When the stannous citrate solution is to be lyophilized, a "fillercompound" may be added to the solution to provide bulk or mass and toaid in the lyophilization process. Good results have been achieved usinglactose as the filler compound.

In one particular embodiment of the invention, an aqueous solution ofstannous citrate was prepared by combining about 75 mg citric acid withabout 750 ug stannous chloride. About 250 ug gentisic acid was added.When 50 ug of gentisic acid was added, the stabilizing effect was not asefficient, whereas 1 mg gentisic acid was found to be too large anamount, having a negative affect on yields. About 100 mg lactose (apreferred amount) is then added to the preparation, although about 20 mgis generally adequate. The final solution (about 2 mLs volume) then islyophilized.

Perrhenate is added to the stannous citrate preparation. Perrhenate canbe introduced into the preparation as an aqueous solution of the sodiumsalt (e.g., eluted from a rhenium generator) or as an aqueous solutionof the tetrabutylammonium ion pair, as described in Example 16 below.Either way, perrhenate is incubated with a solution comprising areducing agent and a complexing agent. The reaction mixture is incubatedat about 25° C. to about 50° C., preferably at about 25° to 37° C., fora minimum of 10 minutes. Incubation for 10 minutes generally givessufficient yields of the desired rhenium exchange complex (e.g.,rhenium-citrate), while minimizing the formation of insoluble rheniumdioxide.

A chelating compound of the invention comprising thioacetal orhemithioacetal sulfur-protecting groups, as described above, isdissolved in an organic solvent effective in dissolving the chelatingcompound and suitable for the exchange reaction that follows. Suitablesolvents should be non-toxic in mammals and inert toward the reactantsin the reaction mixture. Organic solvents which may be used includeacetonitrile, ethyl acetate, and methyl ethyl ketone. When theradiolabeled protein is to be injected into humans, however, suitableorganic solvents include but are not limited to alcohols such asethanol, butanol, t-butyl alcohol, and propanol and polar aproticsolvents such as DMSO and dimethylformamide. The choice of solvent mayvary according to the particular chelating agent included in the kit.For example, when the chelating compound comprises a tetrafluorophenylester group, ethanol will react with the ester in a transesterificationreaction, producing ethyl ester by-products which are undesirablylipophilic and which are much less reactive toward free amine groups onproteins. A preferred organic solvent is isopropyl alcohol.

The solution comprising the chelating compound is combined with therhenium exchange complex solution prepared above to form thecorresponding rhenium chelate compound. The reaction advantageously isconducted at a pH of from about 1.5 to about 5.0, preferably from about1.7 to about 2.0. At these acidic pH conditions, the formation ofinsoluble ReO₂ will be minimized; and as explained above, hemithioacetaland thioacetal sulfur-protecting groups will be displaced by ametal-assisted acid cleavage during the rhenium labeling exchangereaction to form the corresponding rhenium chelate compound. Also,hydrolysis of ester groups on the chelating compound is minimized underacidic conditions when compared to basic conditions. If adjustment ofthe pH of the reaction mixture is necessary, suitable acids may be addedin amounts sufficient to displace the sulfur-protective groups in thepresence of the metal radionuclide (i.e., in amounts sufficient toadjust the reaction mixture to the above-described pH values range).Suitable acids include but are not limited to phosphoric acid, sulfuricacid, nitric acid, glacial acetic acid, hydrochloric acid, andcombinations thereof. Also included are solutions comprising such acidsand buffers (e.g., acetate and phosphate buffers).

The amount of chelating agent reacted with the Re-citrate intermediatemay vary according to the reaction volume, which in turn variesaccording to the volume in which perrhenate was added in an earlier step(e.g., perrhenate may be added as an eluate from the generator or mayfirst be concentrated). In one embodiment of the invention, good resultshave been achieved when the concentration of chelating compound in thereaction mixture (in which the chelate is formed) is about 100 ug toabout 200 ug of chelating compound per mL of reaction mixture.

The reaction mixture is heated between about 50° C. and 100° C. for fromabout 5 to about 45 minutes. Good results have been achieved by heatingat about 75° C. for about 10 minutes. Upon completion of the reaction,the mixture is transferred immediately to a 0° C. ice bath for a minimumof 2 minutes to stop the reaction and minimize the hydrolysis of theester group.

The next step (protein conjugation) may vary according to the volume ofthe reaction mixture in which the chelate was formed, which may varyaccording to the volume of the perrhenate solution added earlier. Whenthe perrhenate was added in a relatively large volume (e.g., about 3 mLsas an eluate from a generator as in Example 16 below), the chelate maybe purified from the chelation reaction mixture using a preparativereversed phase column. Suitable columns include but are not limited toBaker C18 and C8 columns. The desired chelate is retained by the columnpacking material, while most impurities (e.g., starting reagents such ascitric acid, gentisic acid, stannous chloride, and lactose) may bewashed off the column. Good results have been achieved by washing thecolumn (after sample loading) several times with water, then severaltimes with a 2% to 20% ethanol/phosphate buffer solution. The columnthen is dried, and the chelate compound is eluted with an organicsolvent, preferably CH₃ CN, that can be dried off under mild conditions.Usually, a flow of nitrogen dispensed through needles evaporates all thesolvent, leaving a white residue in the elute vial. An aqueous solutioncomprising a buffer is added to the protein to be radiolabeled, which inturn is added to the vial containing the chelate. Sodium bicarbonatebuffers are preferred. The other parameters for the protein conjugationstep are as presented above for the diagnostic kit.

Alternatively, when perrhenate is added to the stannous citratepreparation in a smaller volume (e.g., when the perrhenate has beenconcentrated as a tetrabutylammonium ion pair [see Example 16]), anaqueous solution comprising a buffer is directly added to the chelationreaction mixture to raise the pH to about 5.5 before adding the proteinto be radiolabeled. The choice of the buffer is as presented for thediagnostic kit. The same buffer is added to the polypeptide to beradiolabeled, which in turn is added to the buffered Re-chelate.Sufficient buffer is added to the protein solution so that the final pHof the conjugation reaction is from about 9 to about 11. Theconcentration of the protein and the temperature during conjugation aresimilar to those already presented for the diagnostic kit.

After the protein conjugation step, L-lysine may be added to thereaction mixture to displace the ester containing chelate compound whichmay be associated with (e.g., adsorbed to) but not covalently bound tothe protein. It is believed that reaction of the ester group on thechelate with the free amine group of L-lysine helps displacenon-covalently bound chelate from the protein.

The desired radiolabeled polypeptide (i.e., the chelate-polypeptideconjugate) then is purified from the reaction mixture using any suitablemeans. Good results may be achieved using an anion exchange column(e.g., a DEAE Sephadex or QAE Sephadex column) or a gel permeationcolumn. A QAE-Sephadex column is generally preferred.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1

Synthesis of

N,N'-bis(benzoylmercaptoacetyl)-3,4-diamino Butyrate.

In a dry flask under nitrogen is placed 1.54 g (0.010 mole) of3,4-diaminobutyric acid hydrochloride and 250 mL of absolute ethanol.Dry HCl gas is then bubbled into the solution. The mixture is refluxedfor one to two days until formation of the ethyl ester is complete. Theproduct is then concentrated to a dry solid and the hydrochloride esterdissolved by rapid stirring at ice bath temperature in a mixture of 50mL toluene and 50 mL of saturated sodium bicarbonate. To this solutionis added 5.0 g (0.044 mole) of chloroacetyl chloride in 10 mL of tolueneby dropwise addition. After addition is complete, the mixture is allowedto come to room temperature and stirred for an additional 30 minutes.Layers are separated, and the aqueous portion is extracted twice withethyl acetate. The organic layers are combined, washed with water andbrine, and dried (magnesium sulfate). Removal of the solvent leaves theproduct as a white solid, which may be used without furtherpurification.

A solution of 1.41 g (about 4.45 mmole) of the bischloroacetamide isprepared in 10 mL of dry ethanol under nitrogen. To this is added asolution of sodium thiobenzoate in dry ethanol, prepared from sodiummethoxide (0.204 g of sodium, 8.87 mmole, and ethanol), which is reactedwith 1.23 g (8.90 mmole) of thiobenzoic acid. After a few minutes atroom temperature, precipitation occurs. The reaction is heated to refluxfor 30 minutes. It is then allowed to cool, diluted with ethyl acetate,washed with water and brine and dried (magnesium sulfate). Removal ofsolvent leaves a cream-colored solid which may be recrystallized fromtoluene.

Example 2

Radiolabeling with Tc-99m.

1. The product prepared in Example 1 (0.1 mg) is dissolved in 0.3 mL ofethanol by heating and 30 ul of 5 N sodium hydroxide and 0.3 mL of wateradded in succession. After heating for 15 minutes at 95° C. during whichtime the ethanol evaporated, an essentially aqueous solution of thehydrolyzed ligand is left. To the mixture is then added generatorpertechnetate in saline (0.5 mL or less) which includes about 30 mCi orless of Tc-99m and 0.5 mg of freshly dissolved sodium dithionite; or (2)after allowing the mixture to stand for a short period at roomtemperature, the mixture is heated to 95° C. for an additional 15minutes and the pH adjusted to about 8.

2. The protected thiol, free carboxylic acid ligand of Example 1, 0.10mg, is added to 20 mg of sodium gluconate and 0.010 mg of SnCl₂.2H₂ O,pH adjusted to 5. The Tc-99m as pertechnetate is added to the mixtureand the mixture heated at 95° C. for 5 minutes.

The product mixture may be purified by preparative HPLC, using a 25cmoctadecylsilane column (Altex Model 312 chromatograph; 4.6×250mm ODSultrasphere, 5u) and eluting with 95% 0.01 M sodium phosphate (pH 6) and5% ethanol with a flow rate of 1.0 mL/minute. The preparations areanalyzed for reduced hydrolyzed technetium on silica gel thin-layerstrips.

Example 3

Formation of Activated Esters.

The conditions for formation of the activated esters are as follows:Into a reaction flask is introduced the carboxylic acid ligand or tracerlevel of metal complex carboxylate and an equimolar amount of thehydroxylic compound and a small excess, about 25% excess, of1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (ECDI) and 400ul of dimethylformamide (DMF). Upon completion of the reaction, sodiumacetate is added to quench unreacted ECDI and the solution is ready foruse for conjugation.

The protein to be conjugated is dissolved in 0.2 M borate buffer, pH 8.5to 9.0, to a protein concentration of about 2 to 5 mg/mL. The mixture isallowed to stand at 4° C. until all of the protein has dissolved. To theaqueous protein solution at a pH adjusted to 8.5-9.0 is added the estersolution and the pH readjusted if necessary. The resulting conjugate isthen preparatively chromatographed on an HPLC gel filtration column with0.05 M phosphate, pH 7.4, buffer as eluant.

In the following study, various conditions were employed, employingactivated esters of technetium chelate prepared as described above forreaction with immunoglobulin under varying conditions of time,temperature, concentration and pH. The following Table 1 indicates theresults.

                                      TABLE 1                                     __________________________________________________________________________    Reactions of Activated Esters of N,N'-bis(mercaptoacetyl)-3,4-diaminobutan    oate                                                                          (Tc-99m) with Immunoglobulin under Different Conditions                       Activated Protein     % Labeled                                                                           % Ester                                                                             % Ester                                     Ester  pH mg/ml                                                                             T(°C.)                                                                     t(min)                                                                            Protein                                                                             Hydrolyzed                                                                          Unreacted                                   __________________________________________________________________________    p-nitrophenyl                                                                        6.94                                                                             1.0 23   22  0.5   3.7  95.8                                               6.94                                                                             1.0 23   77  1.7   8.2  90.1                                               6.94                                                                             1.0 37   60  4.3  17.5  78.2                                               6.94                                                                             1.0 37  105  6.4  24.9  68.7                                               8.70                                                                             1.0 23  105 12.0  33.0  68.7                                               8.70                                                                             1.0 34  120 15.0  69.0   8.0                                               8.70                                                                             1.0 23  320 20.0  50.0  23.0                                               9.69                                                                             1.0 23  120 13.0  76.0   7.0                                        2-chloro-4-                                                                          8.55                                                                             1.0 23  240 34.0  62.0   4.0                                        nitrophenyl                                                                          8.55                                                                             1.0  0  240 37.0  60.0   3.0                                               9.22                                                                             1.0 23  300 26.0  72.0   2.0                                               9.22                                                                             1.0  0  330 30.0  66.0   4.0                                               8.61                                                                             3.0 23   30 46.0  39.0  15.0                                               8.61                                                                             3.0  0   60 38.0  46.0  16.0                                               8.61                                                                             3.0 23   75 51.0  38.0  11.0                                               8.61                                                                             3.0   0 100 44.0  42.0  14.0                                        hydroxybenz-                                                                         6.80                                                                             1.0 23   30  8.4  45.4  46.0                                        triazole                                                                             6.80                                                                             1.0 23   60 10.3  48.3  39.0                                               6.80                                                                             1.0 23  120 10.8  53.3  35.8                                               8.50                                                                             1.0 23   90 10.3  61.0  23.0                                               8.70                                                                             1.0  5  120 32.0  30.0  32.0                                               8.70                                                                             1.0  5  240 34.0  30.0  30.0                                               9.25                                                                             1.0  5   45 12.0  33.0  53.0                                               9.25                                                                             1.0  5   90 13.0  32.0  53.0                                               9.40                                                                             1.0 23   60  4.3  66.0  21.0                                        __________________________________________________________________________

Example 4

Synthesis of 4,5-diaminopentanoate.

To a solution of 50.5 g of sodium bicarbonate in 200 mL of water wasadded 85.0 g of glutamic acid gamma-ethyl ester and the mixture cooledin an ice-salt bath. While maintaining the temperature between 0°-5° C.,40 g of carbobenzoxy chloride was added and the mixture stirred for 5hours followed by warming to room temperature and stirring for anadditional 2 hours. After extraction 2×100 mL of ether, the mixture wasacidified with 6 N HCl to Congo red (pH 3). The separated oil wasextracted with 3×100 mL methylene dichloride, the combined organiclayers washed with brine and water and then dried over anhydrous sodiumsulfate. Evaporation and crystallization from 200 mL carbontetrachloride gave a yield of 46.3 g (77%). MP86°-88° C.

To a solution of 46 g of the above product in 45 mL of THF at 35°-40° C.was rapidly added BH₃ -THF (0.18 mmol in 178 mL). After 3 hours, analiquot on TLC (ethyl acetate hexane 4:1) showed substantially completeconversion to the alcohol.

Fifty mL of ethanol was added to the reaction mixture and the mixtureevaporated to dryness. After repeating the procedure twice with 100 mLof ethanol, the residue was suspended in water, extracted with ethylacetate and the organic layer washed successively with 2×100 mL of 2%aqueous bicarbonate and water, followed by drying over anhydrous sodiumsulfate. The organic solvent was then evaporated, the residue dissolvedin hexane and upon cooling gave 30.8 g (71%) yield of a low-meltingsolid. MP86°-88° C.; TLC (Rf ethyl acetate-hexane 0.19).

The alcohol (29.5 g) prepared above was dissolved in 90 mL of pyridine(0° C.-5° C. ) and 19.5 g of tosyl chloride added at once. Precipitationof pyridinium-hydrochloride was observed after 1 hour and the mixturestirred for 2 hours more, followed by storage at 4° C. overnight. Thesolution was poured with stirring into a liter of ice-water and theresulting solid isolated by filtration, washed with water and dried in adesiccator overnight to yield 35 g (80%) of the tosyl ester. MP73°C.-76° C.

To the tosyl ester (22.45 g) in 150 mL of DMF was added 3.9 g of sodiumazide and the mixture heated at 50° C.-55° C. for 3 hours. At the end ofthis time, the DMF was removed in vacuo at 5-10 torr., cold water addedand filtered. The resulting azide was dried in a desiccator overnight toyield 14.56 (91%) of the desired product. MP60° C.-63° C.

Into 227 mL of 1 N HCl-ethanol (abs) was dissolved 14 g of the aboveazide and the solution carefully added to 1.4 g of platinum oxide in ahydrogenation bottle. The mixture was hydrogenated at 50° C.-55° C. for48 hours and the course of the reduction followed by TLC. At completionof the reaction, the catalyst was removed by filtration, the filtrateevaporated to dryness and the residue dissolved in 325 mL of 6 N HCl andthe mixture refluxed for 36 hours. After filtration and evaporation todryness, the residue was dissolved in 100 mL of water, the waterevaporated and the process repeated twice. The residue was trituratedwith ethanol to yield 8.3 g (91%) of the diamino acid product. MP 250°C.

Example 5

Synthesis of Antibody N₂ S₂ Conjugate Using

o-Nitrophenyl Disulfide Protected Ligand.

To 2.05 g of the above diamino acid dissolved in 50 mL of DMF was addedtriethylamine (3 mL) and succinimidyl S-benzoyl thioglycolate (5.86 g)and the mixture stirred for 15 minutes. Dimethylformamide was removed invacuo and 100 mL of cold water was added. The precipitated oilsolidified on standing. The solid was filtered, dried and crystallizedfrom ethyl acetate. MP 126°-127° C.

To sodium ethoxide (140 mg sodium) in 30 mL ethanol was added 0.966 ofthe above product and the mixture stirred overnight at room temperature.After evaporating the solvent in vacuo, the residue was dissolved inglacial acetic acid, the solvent evaporated and the process repeatedtwice. The residue was redissolved in 30 mL of glacial acetic acid and0.77 g of o-nitrophenylsulfenyl chloride added and the mixture stirredat room temperature for 24 hours. The reaction was monitored by TLC(acetonitrile-water 95:5), and at completion of the reaction, the aceticacid was removed in vacuo and cold water added. The solid precipitatewas filtered, washed with cold ethanol (10-15 mL) and dried in vacuo for12 hours over P₂ O₅. The yield was 1.03 g (88%). MP 200° C.TLC:acetonitrile:water 95:5 R_(f) 0.39.

To the bis-(di-o-nitrophenyldisulfide (0.293 g) suspended in 50 mL THF(anhydrous) was added N-hydroxysuccinimide (63 mg) followed bydicyclohexylcarbodiimide (113 mg) and the mixture stirred for 48 hoursat room temperature. The solution was concentrated to about 15-20 mL andcooled, the precipitate removed by filtration and the filtrate dilutedwith 25-30 mL of ethyl acetate, followed by washing the organic layerwith water. The organic layer was dried over magnesium sulfate,concentrated to 20 mL and cooled. The resulting precipitate wasfiltered, the filtrate concentrated to about 10 mL and cooled to about10° C.-15° C. After filtration, the filtrate was maintained at about 4°C. for 2-3 hours. Addition of anhydrous ether to the cold solutionresulted in a yellow precipitate (about 95 mg), followed by a secondcrop of about 90 mg of an impure product.

The antibody conjugation reaction was contained in a final volume of 40mL: 1.8 mg (1.72×10⁻⁵ moles) bis-(di-o-nitrophenyldisulfide) N₂ S₂ligand, 178 mg of mouse monoclonal antibody (IgG, 1.2×10⁻⁶ moles), 4.0mL of redistilled DMF, 0.05 M sodium borate buffer pH 8.5. Afterstirring 90 minutes at room temperature, 4.4 mL of 5 N sodium chlorideand 1.9 mL of 100 mM dithiothreitol were added. After an additional 30minutes the reaction mixture was centrifuged to remove any particulatesand the supernatant fractionated by gel filtration columnchromatography. The column eluent was monitored at 280 nm, and thefractions containing the monomeric antibody conjugate were pooled andconcentrated in an Amicon stirred cell (30,000 molecular weight cutoff).Final yield was 141 mg (82%).

Example 6

Technetium-99m Labeling of Antibody-Ligan Conjugate with Tc-Tartrate.

Stannous tartrate kits were prepared from degassed solutions of 0.5 mLdisodium tartrate (150 mg/mL) and 0.1 mL stannous chloride (1.0 mg/mL inethanol) in an evacuated vial under nitrogen atmosphere. To a stannoustartrate kit, sodium pertechnetate 0.5 mL (about 15 mCi) was added andheated at 50° C. for 10-15 minutes. After cooling to room temperature,quality control for Tc-99m tartrate and insoluble Tc-99m was carried outon Gelman ITLC using methyl ethyl ketone and 0.01 M sodium tartrate pH7.0 eluents, respectively. Tc-99M tartrate formation was typically98-99% with soluble Tc-99m values ranging from 0.1 to 0.2%.

In an evacuated vial, 100 ul saline, 200 ul of sodium phosphate (0.2 M,pH 8.0) and 200 ul of antibody-ligand conjugate (1.9 mg/mL) were addedsuccessively. Immediately after adding the conjugate, 250 ul of Tc-99mtartrate (about 3 to 5 mCi was added and heated at 50° c. for 1 hour.Percent technetium bound to protein and the formation of pertechnetatewere determined by ITLC using 50% MeOH:10% ammonium acetate (1:1) and1-butanol eluents, respectively. Technetium incorporation typicallyranged from 70-88% on a ligand -Ab conjugate with a ligand per antibodyratio of 1.5 to 3.0.

                                      TABLE 2                                     __________________________________________________________________________    Comparative Biodistribution of Tc-99m and Iodine-125                          Anti-melanoma Antibody 9.2.27 in Mice Bearing                                 Melanoma Tumors from FEMX Cell Line.                                          Organ                                                                              Tumor                                                                              Liver                                                                              Spleen                                                                            Lung Stomach                                                                             Thyroid                                                                            Kidney                                     __________________________________________________________________________    Tc-99m                                                                               5.78*                                                                             1.54                                                                               1.34                                                                              1.79                                                                               0.26  0.61                                                                               1.72                                           ±0.32                                                                           ±0.19                                                                           ±0.14                                                                          ±0.67                                                                           ±0.15                                                                            ±0.05                                                                           ±0.12                                   I-125                                                                               3.97                                                                               1.07                                                                               1.59                                                                              1.81                                                                               2.99  7.79                                                                               1.33                                           ±0.61                                                                           ±0.17                                                                           ±0.03                                                                          ±0.12                                                                           ±1.89                                                                            ±4.50                                                                           ±0.04                                   __________________________________________________________________________     Data are mean ±S.D. percent injection dose per gram for three mice at      48 hours post injection.                                                      The method of Hwang, et al., Cancer Res. (1985) 45:4150-4155 was employed                                                                              

Example 7

Labeling of Antibody with Preformed Tc-99m Pentanoyl N₂ S₂ Chelate.

A Tc-99m chelated derivative was conjugated to an antibody as follows.Tc-99m (75 mCi) chelated by N,N'-bismercaptoacetyl 4,5-diaminopentanoicacid was prepared by dithionite reduction of Tc-99m pertechnetate atbasic pH with 25 ug of the N₂ S₂ ligand. The acid was activated byadding the above complex at pH 7 in 0.5 mL water to 100 ul of water:acetonitrile (1:9) containing 3.0 mg of 2,3,5,6-tetrafluorophenol and100 ul of H₂ O:acetonitrile (1:9) containing 7.5 mg of1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (morpho CDI) added. Afterstoring for 18 hours at room temperature, the mixture was purified usinga Baker-10 SPE reversed phase C₁₈ column. The column was conditionedwith 2 mL of ethanol followed by washing with HPLC grade water. Thereaction mixture was then added to the column, the column washed 4 timeswith 2 mL volumes of 10% methanol in 0.01 M sodium phosphate, pH 7.0,and the ester complex eluted with 2.5 mL portions of acetonitrile. Thefirst eluent contained 8.5 mCi and the second 0.18 mCi. The yield was86% after accounting for decay.

To a 2 mL vial was added 4.5 mCi of activated ester complex inacetonitrile, the solvent evaporated in a nitrogen stream and 0.40 mL ofsodium borate (0.5 M, pH 9.0) added. With stirring, 30 1 (9.14 mg/mL) ofantimelanoma antibody (9.2.27) was added. The final proteinconcentration was 0.52 mg/mL. The reaction was followed with TLC usingGelman ITLC SG strips and eluting with 50% aqueous methanol:10% ammoniumacetate (1:1), indicating that 47% protein bound Tc-99m at 15 minutesand 59% at 30 minutes at room temperature. The Tc-99m labeled proteinwas purified by Centricon-10k filter centrifugation. A sample of 92.4%protein bound Tc-99m showed 84.0% binding to FEMX melanoma cells.

Example 8

Preparation of Re-186 4,5-dimercaptoacetamidopentanoylantibody(anti-melanoma antibody 9.2.27).

In an evacuated vial is combined 100 ul of H₂ O, 100 ul acetonitrile,100 ul of citric acid solution (28.8 mg, 1.5×10⁻⁴ mol), 50 ul of ligand(tetrafluorophenyl4,5-di-(tetrahydropyranylmercapto-acetamido)pentanoate (0.40 mg;6.5×10⁻⁷ mol), 50 ul of stannous chloride (0.5 mg, 2.6×10⁻⁶ mol) and 50ul of Re-186 perrhenate in acetonitrile (4.25 ug, 2.3×10⁻⁸ mol). Themixture is heated at 50° C. for 1 hour, and then 0.30 mL of 1 N NaOH isadded.

The tetrafluorophenyl ester product of the Re-186 N₂ S₂ complex ispurified on a C₁₈ Baker-10 SPE column. After application to the column,impurities are washed off with 2×3 mL of H₂ O and 4×3 mL of 10% CH₃OH/.01 M phosphate, pH 7. The product is eluted with 2 mL ofacetonitrile, and then the solution is reduced to dryness under a streamof nitrogen. Yields of product are about 60%.

Conjugation of Re-186 N₂ S₂ complex is done by addition of antibody (160ul of 5 mg/mL) (Morgan, et al., Hybridoma (1981) 1:27), in 340 ul ofborate buffer (0.5 M, pH 9). After 30 minutes at room temperature, 58%of the radioactivity was protein bound. Immunoreactivity determined bybinding of radioactivity to FEMX melanoma cells was 80% after correctionfor nonprotein bound material.

Example 9

Synthesis of Imidate Form of N₂ S₂ Ligand, Conjugation to Antibody andRadiolabeling with Tc-99m.

2,3-(Bis-carbobenzyloxy)diaminopropan-1-ol (2)

A 500 mL hydrogenation bottle was charged with 55 g (0.25 mol) of2,3-dibromopropanol (Aldrich) and 300 mL of 28-30% aqueous NH₄ OHsolution. The mixture was stoppered with an internal thermometer andheated to 75°-85° C. while shaking on a Parr shaker for 23 hours. Whencool, shaking was stopped and the mixture was carefully opened. Themixture was evaporated to a volume of 50 mL by passing N₂ gas through itwhile heating on an oil bath. While hot, 50 mL of EtOH was added and themixture was allowed to cool. The hydrogen bromide salt of2,3-diaminopropan-1-ol was collected by filtration and dried in vacuo toyield 50 g of hard chunks of white solid which was used without furtherpurification.

A solution of 25 g of the crude salt in 110 mL of 4 N NaOH was cooled to0° C. (ice bath), and to the solution was added a solution of 31.4 mL(0.22 mol, 37.5 g) of benzylchloroformate in 100 mL of CH₂ Cl₂. Themixture was stirred rapidly for 30 minutes at 0° C. and 16 hours at roomtemperature. The CH₂ Cl₂ phase was collected, washed with 75 mL ofbrine, dried (MgSO₄), filtered and concentrated. The resulting solid waswashed with 100 mL of Et₂ O, collected by filtration and dried in vacuoto give 10.7 g (24%) of 2 as a white solid which could be recrystallizedfrom CHCl₃ /hexane to give tiny needles. MP 119°-120° C.

2,3-(Bis-carbobenzyloxy)diaminopropyl-1-methanesulfonate (3)

To a suspension of 10.68 g (30 mmol) of 2 and 6.27 mL (4.55 g, 45 mmol)of Et₃ N in 150 mL of CH₂ Cl₂ cooled to 0° C. under N₂ atmosphere wasadded 2.55 mL (3.78 g, 33 mmol) of methanesulfonyl chloride, and themixture was stirred for 30 minutes at 0° C. The resulting clear solutionwas washed successively with 75 mL of 5% HCl, 75 mL of H₂ O, 75 mL of 5%NaHCO₃ and 75 mL of sat. aq. NaCl (all chilled in ice). The CH₂ Cl₂phase was dried (MgSO₄), filtered, concentrated and crystallized fromCHCl₃ /hexane to yield 12.33 g (94%) of white crystals. MP 92-93° C.

3,4-(Bis-carbobenzyloxy)diaminobutyronitrile (4)

A mixture of 6.56 g (15 mmol) of 3, 1.08 g (16.5 mmol) of KCN, 0.40 g(1.5 mmol) of 18-crown-6 and 75 mL of anhydrous acetonitrile (storedover 3A molecular sieves) was refluxed in a nitrogen atmosphere for 19hours. When cool, the mixture was partitioned between 100 mL of 10%NaHCO₃ solution and 200 mL of CH₂ Cl₂. The CH₂ Cl₂ layer was washedsuccessively with 100 mL portions of 5% HCl, water and brine. The CH₂Cl₂ phase was dried (MgSO₄), filtered and concentrated to give 5.47 g ofbrown oil. Two recrystallizations from CHCl₃ /hexane yielded 2.68 g of 4as a white solid. MP 111°-112° C.

3,4-Diaminobutyronitrile dihydrogeniodide salt (5)

To 3.38 g (13.3 mmol) of I₂ in a 100 mL flask under N₂ atmosphere wasadded 5.42 mL (3.87 g, 26.5 mmol) of hexamethyldisilane. The mixture wasimmersed in a 45°-50° C. oil bath until solid I₂ dissolved (30 minutes).The temperature was raised to 100° C. and held for 5 minutes until thecolor disappeared. The solution was cooled to 0° C. with an ice bath anddiluted with 13.3 mL of CH₂ Cl₂. To the 0° C. solution was addeddropwise over 5 minutes a solution of 1.96 g (5.3 mmol) of 4 in 13.3 mLof CH₂ Cl₂. The cooling bath was removed, and the mixture was stirred inthe dark for 3 hours at room temperature. To the mixture was added 2.15mL (1.70 g, 53 mmol) of MeOH, and stirring was continued overnight (16hours). The mixture was cooled to 0° C., and the solid was collected byfiltration and a pH at which the active ester is stable. dried in vacuoto give 1.75 g (100%) of a tan solid 5 which was characterized as itsdibenzoyl derivative.

3,4-Dibenzoylmercaptoacetamidobutyronitrile (6)

To a mixture of 3.27 g (10 mmol) of 5, 7.33 g (25 mmol) ofN-succinimidyl S-benzoylmercaptoacetate and 10 mL of DMF was added at 0°C. under N₂ atmosphere, 3.48 mL (2.52 g, 25 mmol) of triethyl amine. Thecooling bath was removed, and the mixture was stirred for 1 hour. Themixture was diluted with 50 mL of 5% HCl solution and extracted with2×50 mL of CH₂ Cl₂. The combined CH₂ Cl₂ phases were washed with 100 mLof 5% NaHCO₃ solution, dried (MgSO₄), filtered and concentrated in vacuoto yield 6.75 g of a purple tinted solid.

Purification was accomplished by chromatography (silica gel, EtOAc) andcrystallization of purified fractions (CHCl₃ /hexane) to give 3.20 g(70%) of white solid. MP 125°-127° C.

3,4-Bis-methyldithioacetamidobutyronitrile (7)

To a suspension of 455 mg (1.0 mmol) of 6 in 6 mL of EtOH at roomtemperature under N₂ atmosphere was added 2.2 mL of 1 N aqueous NaOH.The mixture was stirred at room temperature for 1.6 hours, and to theresulting clear solution was added 226 ul of methylmethanethiolsulfonate. The mixture was stirred for 3 hours andpartitioned between 20 mL of pH 7 buffer solution and 2×20 mL of CH₂Cl₂. The combined aqueous layers were dried (MgSO₄), filtered andconcentrated to give 591 mg of pink residue. Purification by silica gelchromatography (EtOAc) and crystallization from CHCl₃ /hexane gave atotal of 217 mg (64%) of white amorphous solid 7. MP 121°-123° C.

Methyl 3,4-bis-methyldithioacetamidobutyrimidate hydrogen chloride salt(1)

A suspension of 141 mg (0.41 mmol) of 7 in 1.66 mL of MeOH and 4.15 mLof Et₂ O was cooled to -20° C. (CO₂ /CCl₄), and HCl gas was passedthrough the mixture via septum inlet for 5 minutes, until most of thesolids had dissolved and the solution was saturated with HCl. Themixture was placed in the freezer in a desiccator for 66 hours and thenconcentrated in vacuo to produce a white foamy solid. The solid wasbroken up, washed with three portions of anhydrous Et₂ O, dried in vacuoto give 111 mg (66%) of 1 as an off-white solid which decomposed onheating and also decomposed after several days in a freezer.

Preparation of Antibody Methyl3,4-bis-methyl-dithioacetamidobutyrimidate Conjugate

A 2 mg/mL stock solution of the N₂ S₂ ligand was prepared in dryacetonitrile. The solution was standardized by determining the disulfidecontent using 2-nitro-5-thiosulfobenzoate Thannhauser, et al., Anal.Biochem. (1984) 138:181), and the ligand concentration was found to be5.30 mM.

For conjugation to mouse monoclonal antibody, 0.16 mL of N₂ S₂ligand-acetonitrile solution was added to the reaction vial and thesolvent removed with a stream of dry nitrogen. Antibody (0.62 mL of 8.1mg/mL solution) and 1.0 mL of 0.2 M sodium bicarbonate buffer pH 9.5were mixed and then added to the reaction vessel containing the driedligand. After stirring 30 minutes at room temperature, the entiresolution was added to a fresh vial containing the same amount of driedligand and the solution stirred another 30 minutes. The conjugatedantibody was purified by Sephadex G-25 chromatography in 50 mM sodiumphosphate pH 7.5, 0.5 M sodium chloride. The protein-containingfractions were pooled and concentrated in an Amicon stirred cell to aconcentration of about 2 mg/mL. The solution was made 50 mM inglutathione, stirred 25 minutes, then purified by Sephadex G-25 gelfiltration and concentrated as before. The final solution (1.7 mg/mL)was stored at 4° C. until use.

Radiolabeling of Antibody Methyl3,4-bis-methyldithioacetamidobutyrimidate Conjugate

Tc-99m prepared in a total volume of 1.1 mL of degassed water with 100ug SnCl₂, 9% (v/v) ethanol, 75 mg disodium tartrate and 3.2 mCi sodium(Tc-99m) pertechnetate. The solution was heated at 50° C. for 15minutes. To a separate vial was added 100 ul of the Tc-99m tartratesolution, 100 ul of 0.2 M sodium bicarbonate, pH 10, and 100 ug of theantibody conjugate. The total volume was then adjusted to 0.5 mL with0.15 M sodium chloride and the solution incubated at 50° C. for 60minutes. Analysis by HPLC (TSK column, 0.2 M sodium phosphate pH 7.4,0.15 M sodium chloride) showed 95% of the Tc-99m was associated with theantibody conjugate.

Example 10

Preparation of S-terephthaloyl-substituted N₂ S₂ Ligand.

The mono-tert-butyl ester of terephthalic acid 1 was prepared by themethod of Buckle and Smith, J. Chem. Soc. (1971) 54:2821.

Succinimidyl ester 2 was prepared by stirring 1 with 1.2 molarequivalents of N-hydroxysuccinimide and 1.3 molar equivalents of1,3-dicyclohexylcarbodimide in dry THF at room temperature for 14-16hours. Thin-layer chromatographic analysis indicated the reaction hadgone to completion. The dicyclohexylurea was then removed by filtration,and the resulting liquid was concentrated in vacuo to yield 2 as a whitesolid. Final purification of 2 was accomplished by flash chromatography.

The thioester 3 was prepared by dissolving 1.0 molar equivalents ofmercaptoacetic acid and 2.0 molar equivalents of 4-dimethylaminopyridinein dry THF. The succinimidyl ester 2 was added to the stirring solution.After stirring for 5 hours the reaction was complete as indicated bythin-layer chromatographic analysis. The THF was removed in vacuo, andthe residue was dissolved in CH₂ Cl₂. The solution was then washed withdilute aqueous HCl and dried over anhydrous MgSO₄. Filtration andevaporation of the solvent gave 3 as a colorless oil which solidifiedupon standing.

The succinimidyl ester 4 was prepared by the method of Subramanian (R.F.Schneider, et al., J. Nucl. Med. (1984) 25:223-229).

The carboxylic acid 5 was prepared by dissolving 4,5-diaminopentanoicacid dihydrochloride salt in 1:4 H₂ O:CH₃ CN containing 3.0 molarequivalents of triethylamine and subsequently adding 2.0 molarequivalents of the succinimidyl ester 4. After stirring for 14-18 hoursat room temperature, TLC analysis showed the reaction to be complete andthe solvent was removed in vacuo. The residue was dissolved in ethylacetate and washed with dilute aqueous HCl, water and brine. The ethylacetate layer was then dried over anhydrous Na₂ SO₄. After filtrationand removal of the solvent, a waxy solid was obtained which wasrecrystallized from a mixture of ethyl acetate and hexane to give 5 as awhite solid.

Tetrafluorophenyl ester 6A was prepared by dissolving 5, along with 1.2molar equivalents of 2,3,5,6-tetrafluorophenol in dry THF.1,3-dicyclohexylcarbodiimide (1.2 molar equivalents) was added to themixture, and the mixture was stirred for 12-15 hours. Analysis bythin-layer chromatography indicated the reaction was complete. Thedicyclohexylurea was removed by filtration, and the solvent was removedin vacuo. The residue was purified by flash chromatography to yield theester 6A as a white solid.

Succinimidyl ester 6B was prepared by dissolving 5 along with 1.2 molarequivalents of N-hydroxysuccinimide in dry THF.1,3-dicyclohexylcarbodiimide (1.2 molar equivalents) was added to themixture, and the mixture was stirred at room temperature for 14-18hours. Thin-layer chromatographic analysis indicated the reaction hadgone to completion. The dicyclohexylurea was removed by filtration, andthe solvent was removed in vacuo. The residue was dissolved in ethylacetate and washed with water. The ethyl acetate solution was dried overanhydrous Na₂ SO₄. The drying agent was removed by filtration, and thesolvent was removed in vacuo. The resulting residue was purified byflash chromatography to yield the succinimidyl ester 6B as a whitesolid.

Removal of the tert-butyl protecting groups was accomplished bydissolving the tetrafluorophenyl ester 6A in CH₂ Cl₂ and treating thesolution with excess trifluoroacetic acid, initially at 0° C., thenstirring to room temperature for 3 hours. Thin-layer chromatographicanalysis showed that the reaction had gone to completion. The solventand excess trifluoracetic acid were then removed in vacuo to yield awhite to colorless solid which was recrystallized from CH₃ CN/H₂ O togive 7A as a white powder.

In the case of the succinimidyl ester 6B, the tert-butyl protectinggroups were removed as described above for compound 6A. It wasnecessary, however, to purify the product 7B by flash chromatography.

These reaction sequences were also carried out starting with the monotert-butyl ester of isophthalic acid to obtain the analogous metaisomers of the products described above.

Example 11

Conjugation of N-hydroxysuccinimidyl4,5-diterephthaloylmercaptoacetamidopentanoate to IgG Antibody.

The conjugation was carried out in a total volume of 2.0 mL andcontained 480 ug (7.1×10⁻⁷ moles) N₂ S₂ ligand active ester 6B, 0.2 mLredistilled DMF (10%), 0.15 M sodium chloride, 0.05 M sodium borate, pH8.5, and 10.0 mg mouse monoclonal antibody (6.7×10⁻⁸ moles). Afterstirring 90 minutes at room temperature the reaction was fractionated bygel filtration over Sephadex G-28 in 0.05 M sodium phosphate buffer pH7.5 with 0.15 M sodium chloride. The excluded volume containing theconjugated antibody was collected. To remove any residual nonproteinmaterial, the conjugate was dialyzed 18 hours against 0.05 M sodiumphosphate, pH 7.5, with 0.15 M sodium chloride. Final yield of proteinwas 100%.

Example 12

Tc-99m Labeling of 4,5-diterephthalylmercaptoacetamidopentanoyl-IgGAntibody Conjugate.

To 120 ul saline, 200 ul of 0.2 M sodium phosphate buffer, pH 8, and 80ul of the terephthaloyl sulfur protected N₂ S₂ conjugate (4.66 mg/mL),250 ul of the Tc-99m tartrate (about 4 mCi) prepared as previouslydescribed was added. The reaction mixture was heated at 50° C. for 1hour, which resulted in a Tc-uptake of 90%.

Following the above procedure, the isophthaloyl analog could also beprepared.

It is important that the resulting product provide for maximum formationof the radionuclide conjugates. In addition, there is the concern aboutthe time, since the radioisotopes do decay with time. Thus, by using thecompounds of the subject invention, one can rapidly conjugate proteinsto provide radionuclide-substituted reagents for use in vivo. Thereagents can be provided in pure form, good yield, and the radionuclidemetal is stably maintained as a chelate with the protein for use invivo. Thus, one can safely direct the radionuclide to a desired site,where only low levels of radioactivity will be nonspecifically directedand bound.

Example 13 ##STR13##

Diaminopentanoic Acid.

N-carbobenzyloxyisoglutamine was prepared according to the procedure ofR. Struka and M. Zaoral. Collection of Czechoslav. Chem. Comm. (1977)42:560.

N-Carbobenzyloxyisoglutamine Ethyl Ester (1)

A stirred suspension of carbobenzyloxyisoglutamine (28 g, 100 mmol) andp-toluenesulfonic acid monohydrate (1.9 g, 10 mmol) in 560 mL ofabsolute ethanol was gently refluxed for 12-14 hours or until TLC(1:5:94 HOAc/H₂ /CH₃ CN) indicated that the reaction was complete.

The reaction mixture was concentrated in vacuo and recrystallized fromethyl acetate/hexane to give a white solid: mp 144°-145° C.

N-Carbobenzyloxy-γ-cyano-γ-aminobutyric Acid Ethyl Ester (2)

To a stirred suspension of Cbz-isoglutamine ethyl ester (15.42 g, 50mmol) and pyridine (8.48 mL, 105 mmol) in 360 mL of anhydrous THF at 0°C. was added dropwise a solution of trifluoroacetic anhydride (7.77 mL,55 mmol) in 40 mL of THF, at such a rate to maintain a temperature of0°-5° C. for 1-2 hours or until reaction was complete as evidenced byTLC (5% H₂ O/94% CH₃ CN/1% HOAc; Cu(OAc)₂ stain.

The reaction mixture was concentrated in vacuo to a clear oil. The oilwas taken up in ethyl acetate, washed twice with dilute aqueous Hcl,once with water, once with brine, and dried over Na₂ SO₄. The mixturewas filtered and concentrated in vacuo to a clear oil. Recrystallizationfrom cold ethanol/water gave 11.90 g (82%) of white needles: m.p.61°-62° C.

4,5-Diaminopentanoic Acid Dihydrogen Chloride (3)

A 500 mL Parr Shaker bottle was charged with 3.0 g ofN-carbobenzyloxy-cyano-amino-butyric acid ethyl ester, 500 mg of PtO₂catalyst (Aldrich), 80 mL of EtOH and 80 mL of 6 N HCl. The mixture wasshaken for 16 hours under 50-60 psi H₂ pressure. The mixture wasfiltered and concentrated. The resulting oily residue was dissolved in150 mL of 6 N HCl and heated at 70° C. for 4 hours. The mixture wasconcentrated in vacuo, and to the resulting syrup was added 100 mL ofEtOH. The mixture was allowed to stand in the refrigerator, and theresulting solid was collected by filtration to yield approximately 2 gof 3 as a white powder.

S-(1-ethoxyethyl)mercaptoacetic acid (5a) ##STR14##

A solution of mercaptoacetic acid (17.4 mL, 250 mmol) in 125 mL ofdichloromethane containing p-toluenesulfonic acid monohydrate (0.24 g,1.26 mmol) was cooled to -18°to -25° C. with stirring. Ethyl vinyl ether(23.9 mL, 250 mmol) in 125 mL of dichloromethane was added dropwise tothe cold solution over a period of 90 minutes. The stirring wascontinued for an additional 30 minutes with the temperature maintainedin the -18° to -25° C. range. Then 200 mL of pH=7 phosphate buffer wasadded, and the reaction mixture was allowed to warm with stirring for 10to 15 minutes. The mixture was then poured into a flask containing 900mL of ethyl acetate and 200 mL of water. Layers were separated and theaqueous portion extracted twice with ethyl acetate. The organic layerswere combined, washed with brine and dried (MgSO₄). Removal of thesolvent left 31.4 g of S-(1-ethoxyethyl)mercaptoacetic acid 4 as acolorless oil (77% yield): ¹ H NMR (CDCl₃) 1.15(t,J=7.0Hz, 3H),1.52(d,J=6.4Hz, 3H), 3.36(s,2H), 3.60(m,2H), 4.84(q,J=6.4Hz, 1H),11.65(s,1H). The material was used without further purification.

In similar reactions, mercaptoacetic was reacted with 4b and 4c to give5b and 5c. ##STR15##

S-(Tetrahydropyranyl)mercaptoacetic acid

A solution of mercaptoacetic acid (1.4 mL, 20.0 mmol) in3,4-dihydro-2H-pyran was cooled to 0° c. with stirring. A catalyticamount (20 mg) of p-toluenesulfonic acid monohydrate was cautiouslyadded, and the mixture was allowed to stir at 0° C. for 30 minutes, thento room temperature for 1 hour. The excess 3,4-dihydro-2H-pyran wasremoved in vacuo to leave an oily residue. The residue was dissolved intetrahydrofuran containing 2 mL of 1.0 N aqueous HCl and allowed to stirat room temperature for 20 minutes. The tetrahydrofuran was evaporated,and the residue was dissolved in ethyl acetate. The ethyl acetatesolution was extracted with 5% aqueous sodium bicarbonate. Thebicarbonate extracts were combined and washed with ethyl acetate. Freshethyl acetate was added to bicarbonate extracts, and the aqueous layerwas acidified to pH 1 with 1.0 N aqueous HCl. The layers were separated,and the aqueous portion was extracted twice with ethyl acetate. Theorganic layers were combined and dried (MgSO₄). Removal of the solventafforded 3.28 g of 5b as a viscous oil (93% yield): ¹ H NMR(CDCl₃)1.68(b,6H), 3.34(m,2H), 3.62(m,1H), 3.90(m,1H), 5.05(b,1H), 11.5(s,1H).The material was used without further purification. ##STR16##

S-Methoxymethyl-mercaptoacetic acid (5d)

To a solution of 1.40 mL (1.84 g, 20 mmol) mercaptoacetic acid and 8.36mL (6.07 g, 60 mmol) of triethylamine in 25 mL of DMF at 0° C. was addeddropwise, over 2 minutes, 3.34 mL (3.54 g, 44 mmol) of chloromethylmethyl ether. The mixture was allowed to come to room temperature andstirred for 16 hours. The mixture was partitioned between 50 mL of Et₂ Oand 50 mL of H₂ O. The Et₂ O layer was washed in succession with 50 mLof 5% HCl solution, 50 mL of pH 7 buffer and 50 mL of saturated NaClsolution. The Et₂ O layer was concentrated in vacuo, and the residualoil was dissolved in 20 mL of THF and 2 mL of 6 N HCl solution. Themixture was stirred for 3 hours and partitioned between 50 mL ofsaturated NaCl solution and 50 mL of Et₂ O. The Et₂ O layer was dried(MgSO₄), filtered and concentrated to yield 1.53 g (56%) of 5d which waspure enough to use in the next step: ¹ H NMR (CDCl₃) 3.33(s,2H),3.38(s,3H), 4.72(s,2H), 10.01(brd s,1H).

In a similar manner, 2-methoxyethyl chloromethyl ether (4d) was reactedwith mercaptoacetic acid to give S-(methoxyethoxy)methylmercaptoaceticacid (5d) in 58% yield as an oil.

Succinimidyl S-(1-ethoxyethyl)mercaptoacetate ##STR17##

A solution of S-(1-ethoxyethyl)mercaptoacetic acid (5.76 g, 35.1 mmol)and N-hydroxysuccinimide (4.85 g, 42.1 mmol) was prepared in 100 mL ofanhydrous THF. To this was added a solution of1,3-dicycloherylcarbodiimide (8.70 g, 42.1 mmol) in 65 mL of anhydrousTHF. The mixture was stirred at room temperature for 2 hours or untilTLC analysis indicated complete formation of the succinimidyl ester. Themixture was then filtered, and the filtrate was concentrated in vacuo toa viscous residue. The residue was dissolved in ethyl acetate, washedwith water, brine, and dried (MgSO₄). Removal of the solvent left thecrude succinimidyl ester as an oil, which was further purified by flashchromatography on silica gel, using ethyl acetate-hexanes as the columneluent, to give 5.1 g of S-(1-ethoxyethyl)mercaptoacetic acidsuccinimidyl ester as a colorless oil (56% yield): ¹ H NMR (CDCl₃)1.21(t,J=7.0Hz, 3H), 1.58(d,J=6.4Hz, 3H), 2.83(s,4H), 3.60 (m,4H),4.88(q,J=6.4Hz,1H).

In a similar manner, compounds 6b-6e were prepared. ##STR18##

Synthesis of 4,5-Bis[S-(1-ethoxyethyl)thioacetamido] Pentanoic Acid##STR19##

To a stirring suspension of 4,5-diaminopentanoic acid dihydrochloride(1.64 g, 8.0 mmol) in 32 mL of anhydrous dimethylformamide containingtriethylamine (6.7 mL, 48.0 mmol) was addedS-(1-ethoxyethyl)mercaptoacetic acid succinimidyl ester (4.60 g, 17.6mmol) dissolved in 12 mL of anhydrous dimethylformamide. The reactionmixture was stirred at room temperature for 90 minutes or until TLCanalysis indicated complete formation of4,5-bis[S-(1-ethoxyethyl)thioacetamido] pentanoic acid. Then thereaction mixture was filtered, and the filtrate was concentrated to aviscous oil. The oil was dissolved in ethyl acetate and washed withsuccessive portions of water until no N-hydroxysuccinimide was evidentin the organic phase by TLC. The organic phase was washed with brine anddried (MgSO₄). Removal of solvent afforded 2.0 g of4,5-bis[S-(1-ethoxyethyl)thioacetamido]pentanoic acid as a viscous oilwhich solidified upon trituration with ether (59% yield): ¹ H NMR(CDCl₃) 1.18(t,J=7.2Hz,6H), 1.53(d,J=6.6Hz,6H), 1.88(m,2H),2.45(t,J=6.8Hz,2H), 3.30(s,4H), 3.55(m,6H), 4.10(m,1H),4.77(q,J=6.6Hz,2H), 7.33(m,2H), 9.44(br,1H).

In a similar manner, compounds 7b and 7e were prepared. ##STR20##

Synthesis of2,3,5,6-tetrafluorophenyl-4,5-bis-[S-(1-ethoxyethyl)thioacetamido]pentanoate##STR21##

To a solution of 4,5-bis[S-(1-ethoxyethyl)thioacetamido] pentanoic acid(1.50 g, 3.53 mmol) and 2,3,5,6-tetrafluorophenol (0.88 g, 5.3 mmol) in16 mL of anhydrous tetrahydrofuran was added1,3-dicyclohexylcarbodiimide (0.95 g, 4.6 mmol) with rapid stirring. Themixture was stirred at room temperature for 18 to 24 hours or until TLCanalysis indicated complete conversion to the ester. Then the mixturewas filtered, and the filtrate was concentrated to give a solid. Thesolid was dissolved in a minimal amount of ethyl acetate and allowed tostand at 5° C. for 2 hours. The solution was then filtered to remove anyprecipitated dicyclohexylurea, and the filtrate was concentrated toafford solid2,3,5,6-tetrafluorophenyl-4,5-bis[S-(1-ethoxyethyl)thioacetamido]pentanoate.The solid was washed with ether to remove any remaining2,3,5,6-tetrafluorophenol. After drying in vacuo, 1.64 g of2,35,6-tetrafluorophenyl-4,5-bis[S-(1-ethoxyethyl)thioacetamido]pentanoate was obtained (81% yield). ¹ H NMR (CDCl₃)1.22(t,J=7.2Hz,6H), 1.56(d,J=6.6Hz,6H), 2.06(m,2H), 2.83(t,J=8HZ,2H),3.33(s,4H), 3.60(m,6H), 4.15(m,1H), 4.75(q,J=6.6Hz,2H), 7.22(m,3H).

In a similar manner, the 2,3,5,6-tetrafluorophenyl esters of 7b-7e wereprepared.

The 2-fluorophenyl (8a), 4-fluorophenyl (9a), 2,4-difluorophenyl (10),2-pyrrolidone (11a), succinimidyl (12a), 2,3,5,6-tetrafluorothiophenyl(13a) esters were synthesized by the same method, except that the finalpurification was achieved by flash chromatography.

The N,N-diethylamino ester (14a) was prepared by the establishedisobutylchloroformate mixed anhydride method (The Peptides, Vol. 1, Ch.6, Johannes Meinhofer, Academic Press, 1979 and "The Practice of PeptideSynthesis,"Reactivity and Structure: Concepts in Organic Chemistry, Vol.21, pp. 113-115, Springer-Verlog, 1984).

The cyanomethylester (15a) was also prepared by established method (ThePeptides, Vol. 1, Ch. 6, Johannes Meinhofer, Academic Press, 1979 and"The Practice of Peptide Synthesis,"Reactivity and Structure: Conceptsin Organic Chemistry, Vol. 21, pp. 109-110, Springer-Verlog, 1984).

Compound 16 was synthesized as follows: ##STR22##

A solution of mercaptoacetic acid (13.9 mL, 200 mmol) and p-anisaldehyde(12.2 mL, 100 mmol) was prepared in 250 mL of dichloromethane. To thiswas slowly added boron trifluoride etherate (1.0 mL, 8.1 mmol) at roomtemperature with stirring. The reaction mixture was stirred at roomtemperature for 18 hours, at which point some of the product 16 hadprecipitated. Removal of the solvent left 16 as a white solid. The solidwas collected and washed with portions of dichloromethane. Drying invacuo left 19.1 g of 16 as a white solid (64% yield): ¹ H NMR (d₆ DMSO)3.24(s,2H), 3.30(s,2H), 3.72(s,3H), 5.24(s,1H), 6.82-7.41(m,4H),10.40(b,2H).

Preparation of the bis-succinimidyl Ester (17) ##STR23##

A solution of (4-methoxyphenyl)methanedithiol-S,S'-diacetic acid 16(10.0 g, 33.1 mmole) and N-hydroxysuccinimide (8.37 g, 72.7 mmol) wasprepared in 300 mL of anhydrous tetrahydrufuran. To this was added asolution of 1,3-dicyclohexylcarbodiimide (15.0 g, 72.7 mmole) in 128 mLof anhydrous tetrahydrofuran. After stirring at room temperature forabout 24 hours, the reaction mixture was filtered to remove thedicyclohexylurea by-product of the reaction. Removal of the solvent fromthe filtrate left a white solid. Recrystallization from acetonitrilegave 10.24 g of bis-succinimidyl-(4-methoxyphenyl)methanedithio-S,S'-diacetate 17 (62% yield): ¹ H NMR (d₆ DMSO)2.84(s,8H), 3.74(m,7H), 5.42(s,1H), 7.18(m,4H). ##STR24##

Preparation of (18)

A solution of 4,5-diaminopentanoic acid dihydrochloride (0.601 g, 2.93mmol) in 580 mL of N,N-dimethylformamide and a solution ofbis-succinimidyl-(4-methoxyphenyl) methanedithiol-S,S'-diacetate (1.46g, 2.93 mmol) in 290 mL of N,N-dimethylformamide were addedsimultaneously and dropwise to a solution of triethylamine (0.82 mL,5.88 mmol) in 290 mL of N,N-dimethylformamide with rapid stirring over aperiod of 30 minutes at room temperature. The mixture was then stirredfor 4 hours. Removal of the solvent left an oil which was dissolved inethyl acetate, washed with water and with brine, and dried (MgSO₄).Removal of the solvent left a solid. The solid was triturated with etherand collected by filtration. The solid was washed with ether and driedto give 0.91 g of 18 as a white solid (79% yield): ¹ H NMR (d₆ -DMSO)1.68(m,2H), 2.30(m,2H), 3.20(m,7H), 3.78(s,3H), 5.04(s,1H), 7.20(m,4 H),7.94(b,2H). MS(EI), m/e 398(M⁺), 380(M⁺ -H₂ O).

Preparation of 2,2-propanedithio-S,S'-diacetic Acid (19) ##STR25##

Acid-catalyzed condensation of 2-methoxypropene with mercaptoacetic acidby a method similar to the preparation ofS-(1-ethoxyethyl)mercaptoacetic acid (5a) afforded2,2-propanedithiol-S,S'-diacetic acid as a white crystalline solid inlow yield: ¹ H NMR (d₆ -DMSO) 1.54(s,3H), 3.38(s,2H).

O,O'-bis-succinimidyl(S,S'-isopropylidine)-S,S'-diacetic Acid (20)

To a solution of 897 mg (4.0 mmol) of 19 and 1.01 g (8.8 mmol) ofN-hydroxysuccinimide in 20 mL of THF at 0° C. was added 1.81 g (8.8mmol) of 1,3-dicyclohexylcarbodiimide. The mixture was stirred at 0° C.for 1 hour and at room temperature for 2 hours. The white solids wereremoved by vacuum filtration, and the filtrate was concentrated to awhite solid which was allowed to stand in 20 mL of CH₃ CN overnight. Thesolution was filtered again, and the filtrate was concentrated to givean oily solid. Recrystallization of the oily solid gave 1.22 g (73%) ofa white solid: ¹ H NMR (CDCl₃) 1.74(s,6H), 2.89(s,8H), 3.75(s,4H).

S,S'-acetonyl-4,5-bis(thioacetamido) pentanoic acid (20)

To a solution of 205 mg (1 mmol) of 4,5-diaminopentanoic aciddihydrochloride and 557 ul (405 mg, 4 mmol) of Et3N in 100 mL of DMF wasadded over 40 minutes a solution of 224 mg (1 mmol) of 19 in 100 mL ofDMF. The mixture was stirred for 2 hours and concentrated to a viscousoil in vacuo. Purification by silica gel chromatography yielded an oilwhich was triturated with ether. The resulting white solid was collectedby vacuum filtration to yield 68 mg (21%) of white powder: ¹ H NMR (CD₃OD) 1.78(s,6H), 1.62-2.00(m,2H), 2.14-2.63(m,2H), 3.29(s,4H), 3.00-3.50(m,2H), 3.78-4.35(m.1H), 7.30-8.00(bid,2H).

Example 14

Conjugation of Chelates Comprising Esters to an Antibody.

Chelate compounds of the invention comprising various ester groups wereconjugated to an antibody. The six chelating compounds tested had theformula: ##STR26## wherein E represents the leaving group of an ester,chosen from o-fluorophenyl; p-fluorophenyl; 2,4-difluorophenyl;2,3,5,6-tetrafluorophenyl; cyanomethyl; and N-hydroxypyrrolidone groups.The sulfur-protecting groups shown are ethoxyethyl groups. Thesecompounds were prepared as described above. Each of the six compoundswas radiolabeled, to form the chelate, as follows:

To 100 ul of a solution containing 5 mg of sodium gluconate and 0.1 mgof SnCl₂ in water, 500 ul of ^(99m) TcO₄ -(pertechnetate) was added.After incubation at room temperature for 10 minutes to form aTc-gluconate complex, 100 ul of the chelating compound (dissolved inIPA/AA 90:10, a 1 mg/mL solution), 80 ul of 0.2 N HCl and 200 ul ofisopropyl alcohol were added, in that order. The reaction mixture washeated at 75° C. for 15 minutes, then cooled on ice for 5 minutes. Next,100 ul of 1.0 M sodium bicarbonate buffer or phosphate buffer wereadded, wherein the pH of the added buffer was such that the pH duringthe following protein conjugation step was as shown in Table 3 for eachof the reactions. Next, 400 ul of a 5 mg/mL buffered solution of anantibody was added, followed by addition of 300 ul of the same buffer.The antibody was a monoclonal antibody designated 9.2.27, which isspecific for an antigen which is a 250 kilodaltonglycoprotein/proteoglycan complex associated with human melanoma cells.This monoclonal antibody has been described by Morgan, et al.,Hybridoma, 1:17 (1981). The reaction mixtures were incubated at roomtemperature for either 15 minutes, 30 minutes or 1 hour, as shown inTable 3. The percentage of the ester-containing chelate in each reactionmixture which was conjugated to the antibody was determined by standardinstant thin-layer chromatography (ITLC) in 12% TCA. ITLC is a knownprocedure, generally described in Nuclear Medicine Technology andTechniques, ed. Bernier, D., Longan, J., and Wells, L.; The C.V. MosbyCo., St. Louis, 1981; pp. 172-174. The procedure is described in moredetail in Example 15. The results are shown in TABLE 3.

                                      TABLE 3                                     __________________________________________________________________________            YIELD            % CONJUGATION AT                                             (% PURITY)                                                                            PH DURING                                                                              ROOM TEMPERATURE                                     ESTER   HPLC                                                                              ITLC                                                                              CONJUGATION                                                                            15 MIN                                                                             30 MIN                                                                             1 HR                                       __________________________________________________________________________    o-fluorophenyl                                                                        96  87/22                                                                              9.94    18   17   22                                         p-fluorophenyl                                                                        99  94/10                                                                              9.90    11   16   11                                         2,4-di- 96  93/7                                                                               9.80    13   14   14                                         fluorophenyl                                                                  2,3,5,6-tetra-                                                                        90  86/14                                                                              9.88    49   51   50                                         fluorophenyl                                                                  cyanomethyl                                                                           98  82/14                                                                             10.0     17   15   --                                                 97  --  8.7      19   14   --                                                 98  --  7.5      21   38   --                                                 97  --  6.8       9   10   --                                                 97  --  6.3      20   15   --                                         N-Hydroxy                                                                             88  67  10.0     22   22   --                                         Pyrrolidone                                                                           81  --  8.7      22   22   --                                         (NHP)   88  --  7.7      15   14   --                                         __________________________________________________________________________

Example 15

Diagnostic Kit.

A diagnostic kit containing reagents for preparation of a ^(99m)Tc-radiolabeled protein conjugate was used as follows, and as outlinedin FIG. 1. The procedures were conducted under conditions which ensuredthe sterility of the product (e.g., sterile vials and sterilizedreagents were used where possible, and reagents were transferred usingsterile syringes). All of the reagents, buffers and solutions shown onthe flow chart were components of the kit. Proper shielding was usedonce the radioisotope was introduced.

One mL of sterile water for injection was added to a sterile vialcontaining a stannous gluconate complex (50 mg sodium gluconate and 1.2mg stannous chloride dihydrate, available from Merck Frosst, Canada, indry solid form) and the vial was gently agitated until the contents weredissolved. A sterile insulin syringe was used to inject 0.1 mL of theresulting stannous gluconate solution into an empty sterile vial. Sodiumpertechnetate (0.75 mL, 75-100 mCi, eluted from a ⁹⁹ Mo/⁹⁹ Tc generatorpurchased from DuPont, Mediphysics, Mallinckrodt or E.R. Squibb) wasadded, and the vial was agitated gently to mix the contents, thenincubated at room temperature for 10 minutes to form a ^(99m)Tc-gluconate complex.

In an alternative procedure for providing the ^(99m) Tc-gluconateexchange complex, the kit includes a vial containing a lyophilizedpreparation comprising 5 mg sodium gluconate, 0.12 mg stannous chloridedihydrate, about 0.1 mg gentisic acid as a stabilizer compound, andabout 20 mg lactose as a filler compound. The amount of gentisic acidmay vary, with the stabilizing effect generally increasing up to about0.1 mg. Interference with the desired reactions may occur when about 0.2mg or more gentisic acid is added. The amount of lactose also may vary,with amounts between 20 and 100 mg, for example, being effective inaiding lyophilization. Addition of stabilizer and a filler compound isespecially important when the vial contained these relatively smallamounts of sodium gluconate and stannous chloride (compared to thealternative embodiment above). One mL of sodium pertechnetate (about 100mCi) was added directly to the lyophilized preparation. The vial wasagitated gently to mix the contents, then incubated as described aboveto form the ^(99m) Tc-gluconate complex.

A separate vial containing 0.3 mg of a chelating agent in dry solid formwas prepared by dispensing a solution of 0.3 mg chelating agent inacetonitrile into the vial, then removing the solvent under N₂ gas, andthe resulting vial containing the chelating compound was provided in thekit. To this vial was then added 0.87 mL of 100% isopropyl alcohol, andthe vial was gently shaken for about 2 minutes to completely dissolvethe chelating agent, which was 2,3,5,6-tetrafluorophenyl4,5-bis[S-(1-ethoxyethyl)thioacetamido]pentanoate, the structure ofwhich is represented by the formula in Example 14, when E is a2,3,5,6-tetra-fluorophenyl group. Next, 0.58 mL of this solution of thechelating agent was transferred to a vial containing 0.16 mL of glacialacetic acid/0.2 N HCl (2:14), and the vial was gently agitated. Of thisacidified solution, 0.5 mL was transferred to the vial containing the^(99m) Tc-gluconate complex, prepared above. After gentle agitation tomix, the vial was incubated in a 75° C.±2° C. water bath for 15 minutes,then immediately transferred to a 0° C. ice bath for 2 minutes.

To a separate vial containing 10 mg of the F(ab) fragment of amonoclonal antibody (specific for the above described 250 Kdglycoprotein/proteoglycan melanoma-associated antigen) in 0.5 mL ofphosphate-buffered saline, was added 0.37 mL of 1.0 M sodium bicarbonatebuffer, pH 10.0. the F(ab) fragment was generated by treating themonoclonal antibody with papain according to conventional techniques.The monoclonal antibody, designated NR-ML-05, recognizes an epitope onthe 250 Kd antigen which is different than the epitope recognized by theabove- described monoclonal antibody designated 9.2.27. The vial wasgently agitated.

The vial containing the acidified solution of the ^(99m) Tc-labeledchelate (see above) was removed from the ice bath, 0.1 mL of the sodiumbicarbonate buffer was added, and the vial was agitated to mix.Immediately, the buffered antibody solution (above) was added, gentlyagitated to mix and incubated at room temperature for 20 minutes toallow conjugation of the radiolabeled chelate to the antibody.

A column containing an anion exchanger, either DEAE-Sephadex orQAE-Sephadex, was used to purify the conjugate. The column was preparedunder aseptic conditions as follows. Five 1 mL QAE-Sephadex columns wereconnected end-to-end to form a single column. Alternatively, a single 5mL QAE Sephadex column may be used. The column was washed with 5 mL of37 mM sodium phosphate buffer, pH 6.8. A 1.2u filter (available fromMillipore) was attached to the column, and a 0.2u filter was attached tothe 1.2u filter. A 22-gauge sterile, nonpyrogenic needle was attached tothe 0.2u filter.

The reaction mixture was drawn up into a 3 mL or 5 mL syringe, and anyair bubbles were removed from the solution. After removal of the needle,the syringe was connected to the QAE-Sephadex column on the end oppositethe filters. The needle cap was removed from the 22-gauge needleattached to the filter end of the column and the needle tip was insertedinto a sterile, nonpyrogenic test tube. Slowly, over 2 minutes, thereaction mixture was injected into the column. The eluant collected inthe test tube was discarded. The now empty syringe on top of the columnwas replaced with a 5 mL syringe containing 5 mL of 75 mM (0.45%) sodiumchloride solution (from which air bubbles had been removed). The needleat the other end of the column was inserted aseptically into a sterile,nonpyrogenic 10 mL serum vial. Slowly, over 2 minutes, the NaCl solutionwas injected into the column, and the eluent was collected in the serumvial.

The total radioactivity in the serum vial was measured using a dosecalibrator. In two separate kit preparations, the yield of radiolabeledantibody was 57.2% and 60.9%, respectively; and the yield generallyranges from about 45% to 65%. The contents of the serum vial were drawnup into a sterile, pyrogen-free, 30cc syringe and diluted to a totalvolume of 30 mL with sterile 0.9% NaCl for injection into a humanmelanoma patient. A quality control test was performed on a0.01 mLaliquot before injection by instant thin layer chromatography, asfollows.

Supplies

1. Chromatographic Solvent. Prepare a 12% (w/v) trichloroacetic acid(TCA) in water solution. The solvent can be prepared as a stock reagentand is stable for 30 days when stored at 4° C.

2. Silica Gel Impregnated Glass Fiber Sheets. These are available fromGelman Sciences, Inc., Ann Arbor, Michigan, as ITLC™ SG, 20×20 sheets,Product No. 61886. Pre-cut the strips to a final dimension of 2×10 cm.NOTE: The strips are fragile; use caution during handling. Activate thepre-cut strips according to the manufacturer's instructions. Store theactivated strips after activation according to the manufacturer'sinstructions.

Test Procedure

1. An activated TLC chromatographic strip was carefully removed from astorage container using forceps. Using a lead pencil, the origin wascarefully marked with a lead pencil at approximately 1.2 cm from one endof the strip.

2. A small drop (2-5 uL) of product was spotted at the origin. NOTE: Itis not necessary to dry the spot prior to beginning chromatographicdeveloping.

3. The chromatographic strip was then placed into the developingchamber, with care taken not to immerse the origin into the solventbath.

4. The chromatographic strip was developed, allowing the solvent toascend to about 1 cm from the strip top. The strip was then removed fromthe developing chamber and allowed to dry.

5. The developed chromatographic strip was cut into three sections asillustrated below, and the sections were identified as origin, middleand solvent front. ##STR27##

Using the developing system described above, technetium Tc-99m labeledantimelanoma antibody or fragments thereof remain at the origin, andnonprotein-bound Technetrium-99m labeled material travels with thesolvent front. The middle section of the chromatographic strip may beused to verify complete separation between product and impurity (lessthan 5% of total Technetium-99m activity should be assayed on thissection of the strip).

6. Using a suitable radioactivity counter (e.g., a gamma countercalibrated for ^(99m) Tc), each section of the strip was counted. If aradioactive counter is used, continue counting long enough to determinea statistically significant net count for each strip section.

7. The radiochemical purity (percent Technetium-99m antimelanomaantibody) was calculated using the following formula: ##EQU1##

If the radiochemical purity is less than 85%, the material should not beinjected into a human patient. Using this procedure, radiochemicalpurities generally range from about 90% to 99%. The total amount ofradioactivity also was measured prior to injection. In general, from 10to 30 mCi will be administered to a human patient.

Prior to administering the radiolabeled F(ab) fragment (the diagnosticradiolabeled antibody fragment), an irrelevant antibody and an unlabeledspecific antibody were administered to the patient to improve thediagnostic images, as described above. The irrelevant antibody, providedin a separate vial in the kit, was a whole murine monoclonal antibodydirected against a B-cell lymphoma idiotype. The unlabeled specificantibody, also provided in the kit, was a whole anti-melanoma monoclonalantibody designated NR-ML-05, described above. Both the irrelevantantibody and the unlabeled specific antibody were administered asdescribed in Example 17.

The entire 30 mL sample containing the radiolabeled antibody fragmentwas administered to a patient by intravenous infusion. The infusion wascompleted in from about 5 minutes to about 15 minutes. The antibodyfragment concentration in the sample was 0.33 mg/mL.

Target melanoma sites were detected in the patient. The imagingprocedure, using a gamma camera, was as described in Example 17, inwhich patient No. 8501.35 received a radiolabeled diagnostic antibodyfragment prepared using a kit according to the invention.

Example 16

Therapeutic Kit: Preparation of Re-188 Labeled Conjugates.

A therapeutic kit containing reagents for preparation of a ¹⁸⁸Re-radiolabeled protein conjugate was used as follows, and as outlinedin FIG. 2.

Sodium perrhenate (3 mL, 15 mCi, produced from a W-188/Re-188 researchscale generator) was added to a vial containing a lyophilized mixturecomprising citric acid, 75 mg; stannous chloride, 0.75 mg; gentisicacid, 0.25 mg; and lactose, 100 mg. The vial was agitated gently to mixthe contents, then incubated at room temperature for 10 minutes to forma ¹⁸⁸ Re-citrate exchange complex. To a separate vial containing 0.50 mgof2,3,5,6-tetrafluorophenyl-4,5-bis[S-(1-ethoxyethyl)thioacetamido]pentanoate(a C5 N₂ S₂ chelating agent of the invention comprising ethoxyethylS-protective groups and a 2,3,5,6-tetrafluorophenyl ester group), 0.50mL of isopropyl alcohol was added and the vial was agitated for 2minutes to completely dissolve the chelating agent. Next, 0.30 mL ofthis solution was transferred to the vial containing the ¹⁸⁸ Re-citratecomplex prepared above. After gentle mixing, the vial was incubated in a75° C.±2° C. water bath for 15 minutes, then immediately transferred toa 0° C. ice bath for 2 minutes. The yields of ¹⁸⁸ Re-labeled chelatethen ranged between 75% and 90% as measured by reversed phase C₁₈ HPLCanalysis.

A column containing a C₁₈ reversed phase low-pressure material (BakerC₁₈ cartridges) was used to purify the ¹⁸⁸ Re-labeled chelate. Afterconditioning of the cartridge with ethanol and water, the sample wasloaded and washed with three times 2 mL of water and three times 2 mL of20% ethanol/0.01 M phosphate buffer. The column was then dried in vacuoand eluted with two times 1.0 mL acetonitrile. About 75% of the ¹⁸⁸Re-radioactivity was recovered in greater than 95% purity as the esterchelate compound. The organic solvent was then evaporated under a flowof inert gas.

The chelate was then conjugated to a Fab fragment of a monoclonalantibody specific for the above described 250 Kd antigen on melanomacells. This monoclonal antibody has been designated NR-ML-05 and isspecific for a different epitope on the 250 Kd antigen than the 9.2.27antibody described previously.

A buffered solution of the antibody fragment (5 mg/mL, 0.5 mL) was addedto the purified ¹⁸⁸ Re-labeled chelate, followed by 0.5 mL of 0.5 Mcarbonate/bicarbonate buffer pH 9.50. The reaction was kept at roomtemperature for 15 minutes, then 25 mg of L-lysine, 0.1 mL, was addedand the reaction was pursued at room temperature for 15 minutes more.

A column containing Sephadex G-25 material was used to purify the ¹⁸⁸ Reconjugate. The reaction mixture was loaded on top of the column, and 1.2mL aliquots were collected using PBS buffer to rinse the reaction vialand elute the ¹⁸⁸ Re conjugate in the third and fourth fractions. Thepurity of the ¹⁸⁸ Re conjugate was usually greater than 97% for about 35conjugation yields. The conjugate was then further diluted with PBS, andradioactivity was measured prior to injection into the test animals.

In an alternative procedure for preparing the rhenium chelate, the ¹⁸⁸Re (in the form of ReO₄ ⁻⁻ perrhenate) was concentrated on a reversedphase cartridge as the tetrabutylammonium (TBA) ion pair according tothe procedure described in copending U.S. Pat. application having Ser.No. 802,779. The total perrhenate elution of the generator (about 15mL,in saline) was loaded on the cartridge, which was first conditioned with2 mL of 0.01 M TBA. The cartridge was washed with water and 2.5% CH₃ CN,dried under vacuum, and greater than 98% of the perrhenate was elutedwith 1 mL CH₃ CN, which was dried under a flow of nitrogen. This allowedconcentration of virtually all the activity under conditions that didnot affect the yields of ¹⁸⁸ Re-labeled N₂ S₂ active ester chelates, aspresented below. In this alternative embodiment, a kit comprises 25 mgof citric acid, 0.25 mg of stannous chloride, 0.25 mg of gentisic acidand 100 mg of lactose all in a single vial in lyophilized form. Thislyophilized preparation was reconstituted with 1.0 ml of water, and 0.5mL of this solution was added to the dried perrhenate and, after 10minutes at room temperature, 0.1 mL of a 1.0 mg/mL Ethoxyethyl-N₂ S₂-TFP ligand in isopropyl alcohol was added. This is the same chelatingcompound used in Example 15. The reaction was heated at 75° C. for 10minutes, producing ¹⁸⁸ Re N₂ S₂ -TFP esters in greater than 90% yields.Conjugation of the chelate to a protein is accomplished as describedabove.

Example 17

Imaging of Tumors in Humans.

Antibody fragments radiolabeled with ^(99m) Tc according to the methodof the invention were injected into human patients to detect melanomasites within the body. The antibody fragments used were F(ab')₂, Fab' orFab fragments of one of two monoclonal antibodies specific for the 250Kd glycoprotein/proteoglycan antigen of melanoma cells, as shown inTable 4. The fragments were generated by standard techniques (i.e.,pepsin treatment of the monoclonal antibody to generate the F(ab')₂fragment, papain treatment of the monoclonal antibody to generate theFab fragment, and treatment with a reducing agent such as dithiothreitolto generate the Fab' fragment). The two monoclonal antibodies designated9.2.27 and NR-ML-05 are both directed against the 250 Kd of melanomacells, as described above, but are specific for different epitopes ofthe antigen.

The chelate compound having the formula ##STR28## was prepared by one ofthe methods described herein. For patient number 8501.350, the chelatewas prepared and conjugated to the antibody fragment according to theprocedures outlines in Examples 13 and 15. For the other four patients,the chelate was prepared and conjugated generally as described inExample 7. The resulting radiolabeled antibody fragments were purified,and a quality control test was performed, as described in Example 15.Approximately 40 minutes to 1 hour and 30' prior to infusion of theradiolabeled antibody, each patient received 41 to 50 mg of anirrelevant antibody in 12 to 20 mLs of sterile saline by intravenousinfusion. In addition, each patient received 7.5 mg of anon-radiolabeled specific antibody in 20 mLs of sterile saline byintravenous infusion either simultaneously with, or approximately 5minutes prior to infusion of the radiolabeled specific antibody. Thenonradiolabeled specific antibody was either a whole monoclonal bodyspecific for the 250 Kd antigen on melanoma cells (NR-ML-05 for patientnumber 8501.350 and 9.2.27 for the other patients) or a F(ab')₂ fragmentof such an antibody. The irrelevant antibody was a monoclonal antibodydesignated NR2AD, which is a murine IgG_(2a) immunoglobulin that wasdesigned as an anti-idiotype that bound to a single patient's B-celllymphoma and to no other human tissue.

Into each patient was injected 20 to 30 mLs of sterile saline comprisingthe radiolabeled antibody fragment, by intravenous infusion. Thepatients received from 11.4 mCi to about 30 mCi of ^(99m) Tcradioisotope. The desired upper limit of radioisotope administered is 30mCi, and the minimum for effective imaging of tumors is generally about10 mCi. The total amount of protein in the administered solutions rangedfrom 2.5 mgs to 10 mgs. Imaging by gamma camera was performed at fourtimepoints: immediately following infusion of the radiolabeled antibody,at about 3 hours post infusion, at from 7 to 8 hours post infusion, andat from 19 to 20 hours post infusion. The best images of the targetsites (tumors) were achieved by imaging at from 7 to 8 hours aftercompletion of infusion of the radiolabeled antibody. At the two earliertimepoints, much of the radioactivity was still in the patient's blood;and the amount of radioactivity which had localized in target sites wasgenerally insufficient for good visualization of tumors. Imaging at the19 to 20 hour timepoint (attempted in only 2 of the patients) producedimages which generally were fainter and therefore inferior to those ofthe 7 or 8 hour timepoint due to decay of the ^(99m) Tc radioisotope,which has a halflife of about 6 hours. Melanoma sites, includingmetastases, were detected in each patient. Although some accumulation ofradioactivity in the kidneys was detected during these imagingprocedures, the kidneys generally are not considered to be target sitesin the diagnostic procedures of the invention. In addition, tissuesamples were removed from each patient for biopsy at the timepointsindicated in TABLE 4. The various biopsy samples were analyzed in agamma counter to measure the radioactivity, in terms of counts perminute (cpm), in each biopsy sample. The samples were weighed, and thetotal cpm in each sample was divided by the number of mg in the sampleto give the cpm per mg of tissue. The percentage of the total injecteddose of radioactivity (in cpm) which had localized in each of thevarious tissue types sampled was calculated and is shown in Table 4. Theratio of the radioactivity found in tumor site(s) to the radioactivityfound in the other types of tissue also was calculated. The value in the"percent injected dose per mg" column for the tumor tissue in aparticular patient was divided by the value in the "percent injecteddose per mg" column for each non-tumor tissue sample extracted from thepatient to give the tumor:tissue ratio for each non-tumor tissue sample.The results are presented in TABLE 4.

                  TABLE 4                                                         ______________________________________                                        Tumor Localization of Tc-99m Antimelanoma Antibody                                                         %                                                Treat-                  Time Injected                                                                             Tumor:                                    ment  Patient           Point                                                                              Dose   Tissue                                    Group No.      Tissue   (hrs)                                                                              (per mg)                                                                             Ratio Antibody                            ______________________________________                                        5     8501.080 Fat      23.0 .0007  47.1  (Fab').sub.2                                                                  of 9.2.27                           5     8501.080 Fat and  23.0 .0082  4.0                                                      Connec-                                                                       tive                                                                          Tissue                                                         5     8501.080 S.C.     23.0 .0330                                                           Tumor                                                          5     8501.080 Serum    23.0 .0151  2.1                                       5     8501.080 Skin     23.0 .0025  13.2                                      5     8501.100 Adjacent 27.0 .0028  5.6   (Fab').sub.2                                       Fat                        of 9.2.27                           5     8501.100 Fat      27.0 .0010  15.9                                      5     8501.100 S.C.     27.0 .0159                                                           Tumor                                                          5     8501.100 Serum    27.0 .0118  1.3                                       5     8501.100 Skin     27.0 .0100  1.6                                       6     8501.140 Fat      22.0 .0001  43.0  Fab' of                                                                       9.2.27                              6     8501.140 Serum    22.0 .0010  4.3                                       6     8501.140 Skin     22.0 .0004  11.8                                      6     8501.140 Tissue   22.0 .0005  8.6                                       6     8501.140 Tumor    22.0 .0043                                            8     8501.250 Serum    19.0 .0007  5.6   Fab of                                                                        9.2.27                              8     8501.250 Tumor    23.0 .0039                                            9     8501.350 Serum    23.5 .0012  3.3   Fab of                                                                        NR-ML-                                                                        05                                  9     8501.350 Skin     23.5 .0010  3.9                                       9     8501.350 Tumor    23.5 .0039                                            ______________________________________                                    

Example 18

Preparation of a Chelate Compound Comprising a Thiophenyl Ester Groupand Having the Formula: ##STR29##

Sodium pertechnetate (0.5 mL) was added to a freshly prepared stannousgluconate solution in a vial (0.1 mL containing 5.0 mg gluconic acid and120 mg stannous chloride) at pH 6.1 to 6.3. The reactants were incubatedat room temperature for 10 minutes. To the ^(99m) Tc-gluconate exchangecomplex formed in the vial was added 0.1 mL of a C₅ N₂ S₂ chelatingcompound comprising ethoxyethyl S-protecting groups and a thiophenylactive ether group, designated "ethoxyethyl C₅ N₂ S₂ -thiophenylate"(1.0 mg dissolved in a mixture of isopropanol and glacial acetic acid in9:1 ratio), 55 mL of 0.2 N hydrochloric acid, followed by 0.2 mL ofadditional isopropanol. The vial contents were heated at 75° C. for 15minutes to give 80% by HPLC of the technetium-labeled C₅ N₂ S₂-thiophenylate chelate. The solvent system used for HPLC elution of thethiophenylate epimers is 34% acetonitrile, 0.01 M sodium phosphate pH 6.The precipitate observed with 1.0 mg of ligand was rectified by using 20ug instead. The reactivity of ^(99m) Tc-C₅ N₂ S₂ -thiophenylate waschecked by its reaction with lysine as well as with a Fab antibodyfragment. To 0.2 mL of L-lysine (100 mg dissolved in 1.0 mL of 0.5 Mphosphate buffer pH 10.5) was added 0.1 mL of ^(99m) Tc-C₅ N₂ S₂-thiophenylate, which was then incubated at room temperature. Thedisappearance of all the Tc-C₅ N₂ S₂ -tiophenylate was observed in lessthan 15 minutes as indicated by HPLC (34% CH₃ CN, 0.01 M NaPi, pH 6) andby ITLC in both acetonitrile and 12% TCA. Conjugation of the Tc-C₅ N₂ S₂-thiophenylate with a Fab fragment of monoclonal antibody 9.2.27(described above) was carried out at 1.1 mg/mL using 1.0 M phosphatebuffer with three different pH values, as shown in the following Table5.

                  TABLE 5                                                         ______________________________________                                        Antibody Conjugation Reactions                                                Reaction No.                                                                            pH of Solution during Conjugation                                                            ##STR30##                                            ______________________________________                                        1         6.0           24                                                    2         7.0           25                                                    3         8.0           40                                                    ______________________________________                                    

Example 15

Biodistribution Studies in Mice for ^(99m) Tc-labeled MonoclonalAntibody Fragment.

Antibody fragments radiolabeled with ^(99m) Tc were injected into mice,and biodistribution of the radionuclide protein conjugate was analyzed20 hours after injection according to the method of Hwang, et al.,Cancer Res., 45:4150-4155 (1985). The antibody fragment was a Fabfragment of the above-described monoclonal antibody designed 9.2.27,specific for the 250 Kd antigen of melanoma cells. The results are shownin FIG. 3. The set of data labeled "M" represents data for a proteinconjugate prepared, generally as described in Example 7. The set of datalabeled "K" represents data for a protein conjugate prepared using the"kit approach" as described in Examples 13 and 15. The data arepresented in terms of the percentage of the injected radioactivity pergram of each specified tissue type (FIG. 3A) and the tumor/tissue ratioof injected radioactivity (FIG. 3B). The tissue types represented are asfollows: BL=blood; TA=tail; TU=tumor; SK=skin; MU=muscle; BO=bone;LU=lung; LI=liver; SP=spleen; ST=stomach; TH=thyroid; KI=kidney; andIN=intestine. Melanoma sites (tumors) were effectively identified ineach of the mice studied. The data represent the average for four micein each of the two groups ("M" and "K").

Example 20

Biodistribution Studies for ¹⁸⁸ Re-labeled Monoclonal Antibody Fragment.

A chelate compound having the formula: ##STR31## in which theradionuclide metal is ¹⁸⁸ Thenium, was prepared as described in Example8. The chelate was conjugated to a Fab fragment of a monoclonal antibodyspecific for the 250 Kd glycoprotein/proteoglycan melanoma associatedantigen. The monoclonal antibody is designated NR-ML-05, and the Fabfragment was produced by treatment of the monoclonal antibody withpapain according to conventional techniques. The conjugation step andpurification of the resulting radiolabeled polypeptide were as describedin Example 8. The chelate-polypeptide conjugate was injected intotumor-bearing mice, and biodistribution of the radiolabeled material wasanalyzed 20 hours after injection according to the method of Hwang, etal., Cancer Res. 45:4150-4155 (1985). The results are presented in FIG.4 in which the percentage of the injected dose of radionuclide presentin each of the specified types of tissue (per gram of tissue) is shown,including tumor tissue. The same chelating compound was radiolabeledwith ^(99m) Tc and conjugated to the same Fab fragment (as describedabove) and injected into mice. Biodistribution was detected by the samemethod used for the ¹⁸⁸ Re-labeled conjugate, to provide the comparativedata presented in FIG. 4. The tissues analyzed are as follows: BL=blood,TU=tumor, SK=skin, MU=muscle, BO=bone, LU lung, LI=liver, SP=spleen,ST=stomach, NE=neck (thyroid), KI=kidney, and IN=intestine.

Example 21

Biodistribution studies in mice for various antibody fragmentsradiolabeled with ^(99m) Tc.

^(99m) Tc-labeled C₅ N₂ S₂ chelate compounds were conjugated to variousantibody fragments in accordance with the invention. The resultingchelate-antibody conjugates were injected into nude mice bearing tumors,and biodistribution of the injected radioactivity was analyzed accordingto the method of Hwang, et al., Supra.

FIG. 5 shows biodistribution data for a ^(99m) Tc-labeledchelate-antibody conjugate injected into nude mice bearing coloncarcinoma xenografts (tumors). The antibody was a Fab fragment of amonoclonal antibody designated NR-CE-01, which is specific for anepitope of carcinoembryonic antigen, an antigen specific for varioustypes of cancer cells, including colon carcinoma. Approximately 10 ug(100 uCi) of the conjugate was injected into each mouse, andbiodistribution was analyzed at four timepoints: 1, 4, 16, and 24 hourspost injection. FIG. 5A shows the percentage of the injectedradioactivity localized in each of the specified types of tissue,including tumor tissue, per gram of tissue at each timepoint. Relativeclearance of the radioisotope from non-tumor tissue is demonstrated overtime. The abbreviations for the tissue types are as in Example 19. FIG.5B shows the tumor:tissue ratio of injected radioactivity for each ofthe specified tissue types. The biodistribution data were calculated asdescribed in Example 17, with the data being the average for four micesacrificed at each timepoint.

FIGS. 6A and 6B show biodistribution data for two ^(99m) Tc-C₅ N₂ S₂chelate antibody conjugates injected into nude mice bearing coloncarcinoma xenografts (tumors). One of the conjugates comprised a Fabfragment of an antibody designated NR-LU-10, specific for a 40 kdglycoprotein associated with various types of adenocarcinoma cells ofdifferent histologic origin. The other conjugate comprised a Fab'fragment of an antibody designed L11, specific for carcinoembryonicantigen (but for a different epitope of the antigen than theabove-described NR-CE-01 antibody.) Each mouse received 50 ug (about 100uCi) of one of the conjugates. The mice were sacrificed 20 hours postinjection, and biodistribution data were calculated as described above.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced with the scope of the appended claims.

What is claimed is:
 1. A compound of the formula: ##STR32## wherein: oneof Z¹, Z², Z³, or Z⁴ is ##STR33## and the others are H₂ or ═O; wherein(CH₂)_(m) and (CH₂)_(m) ' are aliphatic groups and m=(1-6) and m'=(1-3)and m+m'=(1-6).W is H₂ or ═NH or ═O, with the proviso that the W bondedto the carbon atom bonded to Y is H₂ when Y is --NH₂ ; n is 0 or 1; T isa removable sulfur protective group, hydrogen, or an alkali metal ion; Yis a leaving group of an active ester that reacts with a polypeptide inan aqueous medium to form an amide or amidine bond, wherein said activeester is a carboxylic ester or an imide ester, --NH₂, --NHNH₂, or apolypeptide of at least two amino acids; X is a bond, methylene, or CHZ⁴; and the A's are the same or different and are hydrogen or lower alkylof from 1 to 3 carbon atoms.
 2. The compound according to claim 1,wherein X is a bond.
 3. The compound according to claim 1, wherein the Wbonded to the carbon atom bonded to Y is ═O and wherein Y is a leavinggroup of an active ester that reacts with a polypeptide in an aqueousmedium to form an amide bond.
 4. The compound according to claim 3,wherein said ester is a 2,3,5,6-tetrafluorophenyl ester.
 5. The compoundaccording to claim 1, wherein X is a bond, Z₃ is ═O, and one of Z₁ or Z₂is ##STR34## and the other is ═O or H₂.
 6. A compound of the formula:##STR35## wherein: each T, when taken together with the sulfur atom tobe protected, defines a hemithioacetal sulfur protective group of theformula: ##STR36## wherein R³ represents a lower alkyl group of fromabout two to about five carbon atoms, R⁴ represents a lower alkyl groupof from one to about three carbon atoms, and R⁵ represents hydrogen or alower alkyl group of from one to about three carbon atoms, and Yrepresents the leaving group of an active ester, wherein said activeester is a carboxylic ester or an imide ester.
 7. A compound of theformula: ##STR37## wherein: one of Z¹, Z², Z³, or Z⁴ is (CH₂)_(n) -CWY,wherein n=1-6, and the others are H₂ or ═O;W is H₂ or ═NH or ═O, withthe proviso that W is H₂ when Y is --NH₂ ; Y is a leaving group of anactive ester that reacts with a polypeptide in an aqueous medium to forman amide or amidine bond, wherein said active ester is a carboxylicester or an imide ester, --NH₂, --NHNH₂, or a polypeptide of at leasttwo amino acids; X is a bond, methylene, or CHZ⁴ ; the A's are the sameor different and are hydrogen or lower alkyl 3 carbon atoms; and each T,when taken together with the sulfur atom to be protected, defines ahemithioacetal sulfur protective group of the formula: ##STR38## whereinR³ represents a lower alkyl group of from about two to about five carbonatoms, and R⁴ represents a lower alkyl group of from one to about threecarbon atoms, and R5 represents hydrogen or a lower alkyl group of fromone to about three carbon atoms.
 8. The compound of claim 7 wherein saidhemithioacetal sulfur protective group is an ethoxyethyl group.
 9. Acompound of the formula: ##STR39##
 10. A compound of the formula:##STR40## wherein: Y represents a leaving group selected from ##STR41##and each T, when taken together with the sulfur atom to be protected,defines a hemithioacetal sulfur protective group of the formula:##STR42## wherein R³ represents a lower alkyl group of from about two toabout five carbon atoms, R⁴ represents a lower alkyl group of from oneto about three carbon atoms, and R⁵ represents hydrogen or a lower alkylgroup of from one to about three carbon atoms.
 11. The compound of claim10, wherein said hemithioacetal sulfur protective group is anethoxyethyl group.
 12. A compound of the formula: ##STR43## wherein: oneof Z¹, Z², Z³, or Z⁴ is --(CH₂)_(n) --CWY, wherein n is 1-3, and theothers are H₂ or ═O;W is ═NH or ═O; Y is the leaving group of an activeester that reacts with a polypeptide in an aqueous medium to form anamide or amidine bond, wherein said active ester is a carboxylic esteror an imide ester; X is a bond, methylene, or CHZ⁴ ; the A's are thesame or different and are hydrogen or noncyclic lower alkyl of from 1 to3 carbon atoms; and each T, when taken together with the sulfur atom tobe protected, defines a hemithioacetal sulfur protective group of theformula: ##STR44## wherein R³ represents a lower alkyl group of fromabout two to about five carbon atoms, R⁴ represents a lower alkyl groupof from one to about three carbon atoms, and R⁵ represents hydrogen or alower alkyl group of from one to about three carbon atoms.